Women are twice as likely to suffer from mood disorders than men. Moreover, a growing body of evidence suggests a reciprocal modulation between sex steroids and the serotonin (5‐HT) system. A previous study from our laboratory has shown that the progesterone metabolites 5β‐pregnane‐3,20‐dione (5β‐DHP) and 5α‐pregnan‐3α‐ol,20‐one (3α,5α‐THP), as well as dehydroepiandrosterone (DHEA), increase the firing activity of dorsal raphe nucleus (DRN) 5‐HT neurones in female rats. The present study was undertaken to assess the effects of these steroids in male rats, as well as the effects of testosterone and 17β‐oestradiol (17β‐E) in both sexes, and finally to evaluate gender differences in the modulation of the 5‐HT neuronal firing activity by these different neuroactive steroids. Male rats were treated i.c.v., for 7 days, with a dose of 50 µg/kg/day of one of the following steroids: progesterone, 5β‐DHP, 3α,5α‐THP, DHEA, testosterone, 17β‐hydroxy‐5α‐androstan‐3‐one (5α‐DHT) and 17β‐E. Some rats also received a 3‐day administration of testosterone (50 µg/kg/day, i.c.v). Females were treated in the same fashion with testosterone and 17β‐E. Extracellular unitary recordings of 5‐HT neurones, obtained in vivo in the DRN of these rats, revealed that testosterone and 17β‐E increased the firing activity of 5‐HT neurones in both males and females. In males, the effect of testosterone could already be seen after 3 days of treatment. Neither castration nor any treatment with other steroids significantly modified the firing rate of male 5‐HT neurones. Taken together with previous findings, the results of the present study indicate both similarities and differences between sexes in the modulation of 5‐HT neurones by some steroids. This could prove important in understanding gender differences in mood disorders.