The role of the a form of estrogen receptor (ER alpha) gene expression in the regulation of testosterone-dependent male reproductive behaviors was investigated using ER knockout mice (ERKO), which are specifically deficient in functional ER alpha, but not ER beta, gene expression. Previous studies in gonadally intact ERKO mice revealed that male aggressive behavior was greatly reduced by the lack of a functional ER alpha gene. In the present study the almost complete suppression of male-typical offensive attacks was further confirmed in ERKO mice that had been singly housed since weaning. Regarding aggression, it was also found that ER alpha gene disruption virtually abolished the propensity to initiate offensive attacks, even though ERKO mice could elicit attacks from resident C57BL/6J mice as wild-type (WT) and heterozygous littermates. Daily injection of testosterone propionate (TP) was completely ineffective in inducing aggressive behavior in gonadectomized ERKO mice, whereas it successfully restored aggression in WT mice. In contrast, male sexual behaviors, mounts and intromissions, were induced by daily injection of TP in both gonadectomized ERKO and WT mice. In addition to TP, dihydrotestosterone propionate (DHTP) was also effective in restoring mounts in ERKO mice, although DHTP was much more potent in WT mice than in ERKO mice. Neither TP nor DHTP, however, ever induced ejaculation in ERKO mice. These results together with previous findings in gonadally intact ERKO mice suggest that ER alpha may be responsible for the regulation by testosterone of consummatory, but not motivational, aspects of male sexual behavior. Finally, ERKO male mice retrieved newborn pups placed in their home cage with similar latencies to males of the two other genotypes. During parental behavior tests, however, a higher percentage of ERKO mice (70%) showed infanticide compared with WT mice (35%). The latter result was interpreted as showing that ER alpha activation by testosterone during the perinatal period may exert a suppressive effect on testosterone-inducible infanticide in adulthood. With respect to three major testosterone-dependent behavioral systems reflecting masculinization, these findings demonstrate three different types of effects due to ER alpha gene disruption.