Bovine follicular fluid (bFF) injected ip in mice during 2 days (65,000 U inhibin/day, 1 U inhibin the activity in 1 micrograms bFF protein) caused a significant decrease in the numbers of A4, intermediate (In), and B spermatogonia to 91%, 74%, and 67% of the control values, respectively. The numbers of undifferentiated spermatogonia remained unchanged. These injections suppressed peripheral FSH levels to 6% of the control values, suggesting that FSH might be the modulator of the effects on spermatogenesis. However, in the Chinese hamster, intratesticular injections of bFF during 4 days (6500 U inhibin/day into one testis) also caused a significant decrease in the numbers of A3. In, B1, and B2 spermatogonia to 86%, 61%, 55%, and 94% of the control values, respectively. Similarly, treatment with a partially purified inhibin preparation from rat Sertoli cell-conditioned medium (rSCCM) during 4 days (Mono Q fraction; 1512 U inhibin/day; 37.8 micrograms protein) caused a significant decrease in the numbers of A3, In, B1, and B2 spermatogonia to 90%, 87%, 66%, and 93% of the control values, respectively. Treatment with a highly purified inhibin preparation from rSCCM during 4 days (30K inhibin; 750 U inhibin/day; 100 ng protein) significantly decreased the numbers of In and B1 spermatogonia to, respectively, 87% and 91% of the control values. These effects were limited to the testis into which the material was injected; the contralateral testis or testes injected with control fluid always showed normal numbers of spermatogonia. This implies that the effects on the seminiferous epithelium are not FSH mediated. Intratesticular injections of bFF or pure inhibin did not affect the number of undifferentiated spermatogonia. However, the Mono Q fraction caused a significant increase in the numbers of undifferentiated spermatogonia in stages IV-VII of the cycle, suggesting the presence of a mitogenic factor for undifferentiated spermatogonia in rSCCM which is not present or is counteracted in bFF. The results suggest that inhibin may have a role in the regulation of spermatogonial development in the adult animal.