It is well known that some psychiatric sequelae exist after CO poisoning, but few animal studies on serotonergic neuronal function after CO exposure have been carried out. We investigated the effects of successive carbon monoxide (CO) exposure (6000 ppm, 10 min, 3 repetitions) on serotonergic neuronal systems in rat brain. Serotonin (5-HT) concentrations were significantly decreased only in the frontal cortex from 1 hr to 7 days after CO exposure. 5-Hydroxyindoleacetic acid (5-HIAA) concentrations were significantly increased at 1 hr in all six brain regions measured (frontal cortex, striatum, hypothalamus, hippocampus, midbrain, and pons). 5-HT synthesis, measured by the accumulation of 5-hydroxytryptophan (5-HTP) after the administration of m-hydroxybenzylhydrazine (NSD-1015), was significantly decreased in all regions from 1 hr to 7 days after CO exposure. [3H]Ketanserin (5-HT2A) binding sites in the frontal cortex were not affected by CO exposure. DOI-induced head shakes, a 5-HT2A receptor mediated behavior, were not changed after CO exposure. These findings indicated that CO exposure caused presynaptic serotonergic neuronal dysfunctions that consisted mainly of decreased concentration of 5-HT in the frontal cortex or a decrease of 5-HT synthesis in all six regions, without compensatory hyperfunction of 5-HT2A receptors.