Inducible nitric oxide synthase (iNOS) through its product, nitric oxide (NO), may contribute to the induction of germ cell apoptosis. Using adult iNOS deficient mice, we characterized the reproductive hormonal profile and the testicular phenotype. Although there was no difference in body weight, mean testis weights in mutant mice were 30.77% higher and testicular sperm count 65.51% higher than control animals. No significant differences were apparent in plasma LH, FSH and testosterone levels between these mice. Compared with wild-type mice, histomorphometric analysis showed that the mutant mice had 39.63% increase in the number of pachytene spermatocytes and 33.79% in round spermatids with no apparent changes in the number of preleptotene spermatocytes and spermatogonia. The incidence of spontaneous germ cell apoptosis detected by TUNEL assay was lower at stages I-IV and XI-XII of iNOS mutant mice compared with wild-type animals. The rate of germ cell proliferation estimated by quantitative assessment of the BrdU labeled preleptotene spermatocytes showed no significant change between wild-type and the iNOS deficient mice. When applying testicular warming (43 C for 15 min) to mice, the rate of germ cell apoptosis was elevated predominately at early (I-IV) and late (XI-XII) stages, and less during stages V-VI, VII-VIII and IX-X at 2 and 6 h after heat exposure in the wild-type mice. In contrast, the rate of apoptosis in mutant mice was markedly decreased at early and late stages 2 and 6 h after heat exposure. Pachytene spermatocytes and early round spmeratids were most susceptible to heat-induced apoptosis in both mutant and control animals. Our studies demonstrate that: 1) deficiency of iNOS results in failure to eliminate a small portion of pachytene spermatocytes and round spermatids by apoptosis resulting in a remarkable increase in testis weight and sperm output; 2) deficiency of iNOS confers partial resistance to heat-induced germ cell apoptosis. These experiments suggest that iNOS plays a physiologic role in regulation of germ cell number and in determining testicular size.