This study investigated pubertal changes in neural and behavioral responses to estradiol. Gonadectomized pre- and postpubertal male hamsters (Mesocricetus auratus) were treated with 0.00, 0.05, 0.10, or 0.25 mg estradiol and tested 1 week later for sexual behavior with a receptive female. Estradiol activated behavior in postpubertal, but not prepubertal, males. In contrast, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) immunoreactivity in forebrain nuclei that mediate mating behavior was similar in pre- and postpubertal males. Thus, absence of a behavioral response before puberty is not associated with reduced levels of steroid receptors. Because estradiol induced PR in prepubertal males, these data also suggest that ERa is functional before puberty. Therefore, gonadal steroids facilitate male reproductive behavior only after as-yet-unidentified developmental processes occur during puberty.