Osmoregulatory Ca2+ signaling in hypotonic solutions was studied with videometric techniques in 158 proximal renal tubules isolated from the teleost Carassius auratus. Absence of extracellular Ca2+, hypoxia (23 mmHg), or NaCN (3 mM) did not alter regulatory volume decreases (RVD). Nevertheless, decrements of intracellular Ca2+ via the A23187 ionophore or after intracellular Ca2+ chelation with indo-1/AM (5 microM) inhibited RVD. In tubules depleted of Ca2+, RVD could only be fully elicited when intracellular Ca2+ pulses were given within 1 min after hypotonic stimulation. While inhibition of Ca2+ release from the endoplasmic reticulum (ER) with 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 50 microM) blunted RVD, some of its effects could be reversed with the anion carrier tributyltin (1 microM). Dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP, 0.5 and 1.0 mM) and forskolin (0.25 mM) also impeded RVD; however, their effects could be partially reversed with the K+ ionophore gramicidin (0.5 microM). In conclusion, in Carassius auratus proximal renal tubule cells, RVD is activated by an intracellular Ca2+ signal that likely emanates from the ER and not from the extracellular media or the mitochondrial Ca2+ pool. Ca2+ activation of a cAMP-modulated osmoregulatory K+ channel appears to play an important role.