Sex differences in the brain and behaviour are mostly a result of transient increases in testosterone during the perinatal period. Testosterone influences brain development primarily through aromatization to oestradiol and subsequent binding to oestrogen receptors. Although some studies report that steroid hormones regulate the expression of the inducible transcription factor, Fos, in developing brain, it is not known if there is a sex difference in Fos expression. Changes in Fos protein can be used as an indicator of neuronal/genomic activity. Thus, it provides a useful tool to identify brain regions responding directly or indirectly to steroid hormones. In a first experiment, we examined Fos protein expression in the developing male and female rat brain using western immunoblotting. Dissections were taken from male and female rat pups on the day of birth (postnatal day 0; PN 0), PN1, PN5, PN11 or PN20. Although there was no difference on PN 0, males expressed significantly greater levels of Fos protein on PN1, PN5 and PN20. In a second experiment, we localized the sex difference in Fos protein expression using immunocytochemistry. We found that males expressed significantly higher levels of Fos within a variety of brain regions. These data indicate a sex difference in Fos protein expression during brain development, suggesting a potential role for Fos in differentiating male from female rat brain.