Adult male rats were injected s.c. with either saline, 100 IU hCG, 100 micrograms FSH, 50 micrograms LH, 100 micrograms PRL, 50 micrograms estradiol-17 beta, 500 micrograms or 10 mg testosterone; 50 micrograms estradiol-17 beta; animals were sacrificed at 12-120 h post-injection. Collagenase-dispersed interstitial cells (150-200 X 10(6) cells/2 ml) were incubated in vitro with 10 microCi [3H-methyl]thymidine for 1 h at 32 degrees C. Centrifugation of the cells on discontinuous 11-27% metrizamide gradients revealed thymidine incorporation in the regions of population I and II Leydig cells. A significant increase in thymidine incorporation into DNA after treatment with either hCG or LH was first detectable at 48 h, was equivalent to control values at 72 h and was again significantly increased at 96 h in population I and at 120 h in population II cells. [3H]Thymidine incorporation at 48 h, expressed as dpm/10(6) cells, was 2205 +/- 432 and 4119 +/- 929 vs. 16473 +/- 3795 and 11648 +/- 3427 for control and hCG-treated population I and II cells, respectively. Addition of 20 mM hydroxyurea suppressed [3H]thymidine incorporation, 97% and 96% in hCG-treated population I and II cells, respectively. Autoradiographic analyses revealed that nuclei from control and 48 h hCG-treated population I and II cells exhibited 1.2% and 2.3% vs. 7% and 6.8% silver grains, respectively. PRL had no influence on LH/hCG-enhanced DNA synthesis; however, estradiol-17 beta administration for 48 h dramatically suppressed thymidine incorporation. Population I Leydig cells exhibited a higher level of LH/hCG-stimulated DNA synthesis compared to population II cells.(ABSTRACT TRUNCATED AT 250 WORDS)