The effects of various concentrations of serotonin, ACTH, K
+
, angiotensin II (A ll), angiotensin III (AIII) and [Sar
1
] angiotensin II (SAII) on steroidogenesis and the incorporation of
32
P (after preincubation to near equilibrium with the ATP pool) into phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylcholine (PC) in a preparation of capsular cells from rat adrenals, consisting of 95% zona glomerulosa (z.g.) and 5 % zona fasciculata plus reticularis (z.f.r.) cells, were investigated. Serotonin and ACTH stimulated steroidogenesis in the usual manner but had little or no effect on
32
P incorporation into any of the three phospholipids. However, All, AIII and SAII stimulated steroidogenesis and also
32
P incorporation into PA and PI (maximally to about 280 % of control values) but not into PC. These results taken together with other data on effects on the cAMP output and Ca
2+
fluxes of z.g. cells suggest that stimulation by ACTH and serotonin is mediated by cAMP as second messenger. However, the angiotensins probably act through Ca
2+
, with associated changes in phospholipid metabolism. The
32
P incorporation into PA as a function of lg concentration of All was linear and showed a reasonable index of precision (0.36 + 0.03, eight experiments, 0.23 + 0.02 for a further eight experiments) and correlation with steroidogenesis. The corresponding incorporation into PI showed a maximum effect and a much poorer index of precision (1.02 + 0.30 (4.69 + 3.7)) over the same full range of All concentration used. The effects of AIII and SAII showed similar characteristics for
32
P incorporation into both PA and PI, but, as for stimulation of steroidogenesis, at higher concentrations for AIII than for All. The effects of different doses of All, AIII and ACTH on the corticosterone output and
32
P incorporation into PA, PI and PC of a preparation of cells, consisting of more than 98% z.f.r. cells, from rat decapsulated adrenals were also studied. ACTH, at low doses, which nevertheless markedly stimulated corticosterone output, had a small (maximally to about 125% of control values) but significant effect on
32
P incorporation into PA, PI and PC. The maximum effect was usually at about 10
-10
M ACTH and was not significant at 10
-8
M . All had no significant effect on corticosterone output or on
32
P incorporation into PC. However, All had a significant effect (to about 185 %) on
32
P incorporation into PI, but a smaller effect on incorporation into PA. AII I gave similar results. Increased K
+
concentration (from basal 3.6 to 5.9, 8.4 and 13 mM) stimulated the steroidogenesis of capsular cells but did not increase the
32
P incorporation into PA at any concentration. At 8.4 mM, but not at any other K
+
concentration, there was a small (about 130%) but significant increase in
32
P incorporation into PI. This was not due to incorporation into PI in the contaminating cells as K
+
(or serotonin) did not alter incorporation into PA or PI in z.f.r. cells. Nevertheless, K
+
does not seem to stimulate steroidogenesis by the same mechanism as do the angiotensins, that is, it uses cAMP rather than Ca
2+
as messenger. The results for all the stimuli studied indicate that for a ‘Ca
2+
messenger’ bioassay using adrenal capsular cells, it will be preferable to measure the incorporation of
32
P into PA rather than into PI and this should provide a satisfactory assay as regards specificity, sensitivity and precision.