The oxidative deamination of serotonin (5‐HT) to 5‐hydroxyindoleacetic acid (5‐HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5‐HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5‐HT concentration over the tested range of 5 ± 10⁻⁷ to 10⁻⁴M. At 5 ± 10⁻⁷M 5‐HT, intracellular 5‐HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5‐HT taken up into astrocytes is not stored for re‐release, but is rapidly metabolized to 5‐HIAA, which is then extruded from the cell. At 5 ± 10⁻⁷M 5‐HT, 5‐HIAA formation in intact cells was blocked 63% by the selective high‐affinity 5‐HT uptake inhibitor fluoxetine. 5‐HT oxidation to 5‐HIAA is carried out principally by MAO‐A, because clorgyline was more effective at inhibiting the production of 5‐HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000‐fold more effective than pargyline at inhibiting MAO activity toward ¹⁴C‐labelled 5‐HT. However, the relatively selective MAO‐B substrate β‐phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO‐B activity; the K_m values for MAO‐A oxidation of 5‐HT and MAO‐B oxidation of PEA were 135 and 45 μM, and V_max values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (>20 γM) were oxidized by both MAO‐A and MAO‐B isozymes. These data support the idea that astrocytes in situ might take up 5‐HT and metabolize it to 5‐HIAA using the MAO‐A isozyme. However, sufficient activity of the MAO‐B isozyme appears to be present in astrocytes for metabolism of amines which also show MAO‐B selectivity.