The effects of progesterone (P) on serotonin (5HT) overflow in the ventromedial hypothalamus (VMH), preoptic area (POA) and midbrain central grey (MCG) were studied using in vivo microdialysis. Ovariectomized rats, pretreated with 5 micrograms estradiol, were anesthetized with chloral hydrate and stereotaxically implanted with dialysis probes directed towards one of the respective brain sites. Extracellular 5HT levels stabilized 3 to 5 h following probe implantation. Under stable baseline conditions, perfusion of 1 microM tetrodotoxin through the dialysis probe resulted in 60-65% reduction in 5HT overflow in the brain areas studied. In experiments testing the effect of P on 5HT overflow, rats were subcutaneously injected with 0.5 mg P or propylene glycol vehicle. Samples were analyzed for 5HT at 20 min intervals for 4 h after treatment. Perfusate levels of 5HT were not significantly changed in the VMH, POA or MCG in vehicle-treated rats. Similarly, P treatment failed to significantly alter 5HT overflow in the POA. In the VMH, perfusate levels of 5HT were significantly reduced 60 min after P treatment. Decreases in perfusate 5HT levels were detected 20 min after P in the MCG. The decreases in 5HT overflow measured in the VMH and MCG following P treatment persisted for the remainder of the sampling period with the exception of 1 time point in the VMH. The results provide in vivo evidence for P-influenced decreases in 5HT release in the VMH and MCG. The rapid decrease in extracellular 5HT in the MCG suggests that this effect may represent a non-genomic action of P. These results are discussed in relation to the role of 5HT in the regulation of lordosis behavior.