The regulation of extracellular levels of 5-hydroxytryptamine (serotonin) (5-HT) in the striatum and ventral hippocampus was studied using in vivo microdialysis in awake, unrestrained wild-type 5-HT(1A) and 5-HT(1B) receptor knockout mice. Systemic administration of the selective serotonin reuptake inhibitor fluoxetine evoked a significant dose-dependent increase in extracellular 5-HT in both the striatum and hippocampus at both 2.5 mg/kg (i.p.) and 20 mg/kg (i.p.) in wild-type mice. In 5-HT(1A) receptor knockout mice, the response to 2.5 mg/kg fluoxetine was significantly augmented in the striatum but not the hippocampus, whereas the response to 20 mg/kg fluoxetine was significantly greater in both brain regions. In 5-HT(1B) receptor knockout mice, the increase of extracellular 5-HT was augmented in the hippocampus but not the striatum at both doses of fluoxetine. The response pattern to fluoxetine alone in 5-HT receptor mutant mice corresponded with the effects of fluoxetine given with either the 5-HT(1A) receptor antagonist WAY 100635 (0.1 mg/kg i.p.) or the 5-HT(1B/1D) receptor antagonist GR 127935 (0.056 mg/kg) in wild-type mice. These results indicate common topographical regulation of 5-HT release in different brain regions by genetic mutation and pharmacological challenges. The 5-HT(1A) autoreceptor plays a larger role in regulating 5-HT release in the striatum and possibly other brain regions innervated by the dorsal raphe nucleus, whereas the role of the 5-HT(1B) receptor is relatively greater in the hippocampus and possibly other brain regions innervated by the median raphe nucleus.