Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. Testis weights decreased significantly as early as 1 wk after treatment, with the greatest decrease reached 3-4 wk after treatment, followed by recovery to normal levels 10-15 wk after treatment. We conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60 degrees C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Sperm motility decreased most from treatment of PLSG and SG. After MC or ENU treatment, the greatest loss of acrosin activity and of the acrosin protein was also noted in sperm derived from treated PLSG and SG. MC and ENU failed to induce SDH activity in single sperm resistant to 60 degrees C heat inhibition or to inhibition by malonate. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.