Gender differences in serotonin (5-HT) metabolism, synthesis, and release suggest that the gonadal steroids estrogen (E) and progesterone (P) influence 5-HT neurotransmission. Based on the effects of ovarian steroids in forebrain sites, this might involve changes in the strength of GABA afferent tone in the dorsal raphe nucleus (DRN). To test this hypothesis, the present study used in vivo microdialysis to measure basal extracellular 5-HT in the rat DRN across the estrous cycle and changes in 5-HT in response to the GABA(A) receptor antagonists bicuculline (50 microM) and picrotoxin (50 microM). During proestrus and estrus, baseline 5-HT levels were significantly higher compared to ovariectomized (OVX) rats. Mean baseline levels across experiments were 8.4 +/- 1.3 pg/ 30 microl in proestrus, 5.2 +/- 0.8 pg/30 mul in estrus, and 3.1 +/- 0.5 pg/30 microl in the DRN of OVX rats. Bicuculline and picrotoxin produced significantly greater increases in 5-HT during proestrus compared to estrus. Moreover, in the DRN of OVX rats, bicuculline and picrotoxin produced negligible increases in 5-HT. These data provide evidence of decreased 5-HT efflux and GABA tone in the rat DRN associated with low circulating E and P.