The response of the chromosomes in root-tip cells of
Vicia faba
seedlings has been studied following the exposure of roots to 30 min combined treatments with [
3
H]thymidine and 10
-6
M nitrogen mustard (HN2). Autoradiographs were prepared from squash preparations of root-tips sampled up to 3 days after exposure and metaphase cells were scored for the presence of labelling and for chromosomal aberrations. Only chromatid-type aberrations were induced by HN2 treatment; chromosome-type aberrations characteristic of cells exposed to ionizing radiations whilst in the pre-DNA synthesis (
G
1
) phase of interphase were not observed. At the first mitosis following treatment chromatid structural changes were observed in cells that were exposed to HN2 whilst in the
G
1
and DNA synthesis (
S
) phases of inter-phase, but no aberrations were found in cells exposed whilst in the post-DNA synthesis (
G
2
) phase. In the second mitosis following treatment chromatid-type aberrations were observed in cells that were originally exposed to HN2 whilst in the
G
2
phase and were visibly undamaged at the first mitosis. The frequency of aberrations observed at the second mitosis in cells that were treated whilst in
S
or
G
2
was significantly higher than in
S
cells observed at the first mitosis. From these changes in response with cell phase and from the type of aberrations produced, it was concluded that although HN2 produces lesions in chromosomes independently of cell phase at the time of treatment, structural changes are only produced when the cells undergo DNA synthesis. The nature of the lesions is discussed and it is suggested that chromatid aberrations arise as a result of misreplication of DNA at the sites of alkylation, and that a proportion of the original lesions may be repaired. It is concluded that the high frequency of intrachange relative to interchange aberrations and the localization of aberrations to the heterochromatic regions of the chromosomes are consequences of the requirement for simultaneous replication in chromosome regions that are involved in aberration. The results are compared with those obtained in X-irradiated cells and the relative frequencies of intrachange types induced by HN2 are shown to be similar to the frequencies observed after X-irradiation of
S
cells but not of
G
2
cells. However, the intrachange frequencies do not conform with those predicted on Revell’s exchange hypothesis for aberration formation and it is suggested that this lack of agreement is due to the formation in
S
cells of some iso-chromatid aberrations through a process of misreplication at a single lesion.