The aim of this study was to investigate the turnover of Leydig cells and other interstitial cells in the adult rat testis. Normal adult rats received injections of [3H]thymidine at 9:00 and 21:00 for 2, 5, or 8 days. The percentage of labeled Leydig cells, which was initially low (0.8% +/- 0.2%), gradually increased during treatment to 1.4% +/- 0.3%. The percentage of labeled peritubular cells was considerably higher and increased from 1.4% +/- 0.1% to 3.5% +/- 0.4% during [3H]thymidine treatment. The remaining interstitial cells were the most actively proliferating cells: the percentage of labeled cells increased from 2.4% +/- 0.2% to 7.5% +/- 0.8% during [3H]thymidine treatment. Leydig cells, peritubular cells, and the remaining interstitial cells in the adult rat testis undergo proliferation. By means of a linear regression analysis and an arcsin transformation, an estimation of the time interval needed to replace various types of interstitial cells was obtained. Taking into account the 95% confidence interval, the turnover time of Leydig cells ranged from 142 to 2823 days. The calculated turnover time for the peritubular cells was 85 to 257 days.