Both reduced endogenous estrogen and hemicastration stimulate proliferation of porcine Sertoli cells. The objective of these experiments was to compare the temporal patterns of response to each stimulus with the response to the combined stimuli as indications of shared or separate mechanisms. Within a replicate, one littermate was treated weekly with canola oil vehicle and remained intact; a second littermate was treated weekly with vehicle, and one testis was removed at Day 8; a third littermate was treated weekly with the aromatase inhibitor letrozole to reduce endogenous estrogens and remained intact; and the fourth littermate was treated weekly with letrozole, and one testis was removed at Day 8. Four replicates were evaluated at 2 wk of age, five replicates evaluated at 6.5 wk of age, and five replicates were evaluated at 11 wk of age, with treatment ceasing at 6 wk of age. Numbers of Sertoli cells were determined following GATA4 labeling using the optical dissector method. Levels of estradiol, estrogen conjugates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin were determined by radioimmunoassay. Hemicastration appeared to have a rapid effect on Sertoli cell proliferation, but letrozole treatment had no apparent effect on Sertoli cell numbers at 2 wk of age. Both letrozole treatment and hemicastration had stimulated Sertoli cell proliferation by 6.5 wk of age, although the magnitude of the hemicastration response was much greater. Letrozole appeared to have minimal interaction with hemicastration at this age. Letrozole and hemicastration together increased Sertoli cell numbers at 11 wk of age compared with either treatment alone. Estradiol and estrogen conjugates were dramatically reduced by aromatase inhibition as anticipated; treatment-induced changes in inhibin, LH, or FSH were minimal. Differences in timing of response and positive interaction at 11 wk of age suggest that hemicastration and letrozole stimulate proliferation of Sertoli cells by two initially different pathways.