Apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid-binding proteins, exhibits abundant expression within the CNS of many species, including humans; however, its physiological role remains unclear. Treatment with atypical antipsychotic drugs, especially clozapine, results in elevation of apoD expression levels in rodent brain and in human plasma samples. In order to further explore the role of apoD in mechanisms of clozapine function, we have measured a panel of membrane fatty acids and membrane lipids in brain from drug-treated apoD knock-out mice. Mice received clozapine (10 mg/kg/day) in their drinking water for 28 days and forebrain samples were analyzed using high performance liquid chromatography and capillary gas chromatography. We identified significant differences in the levels of membrane fatty acids in response to clozapine treatment specifically in the brains of apoD knock-out mice, but not wild-type (wt) mice. The most striking observations were decreases in the levels of fatty acids related to metabolism of arachidonic acid (AA), which is a known binding partner for apoD. These include the precursor to arachidonic acid, linoleic acid (LA; 18:2n6c), arachidonic acid itself (20:4n6) and the elongation product of arachidonic acid, adrenic acid (22:4n6). We further report increases in LA, eicosadienoic acid and docosahexaenoic acid in apoD knock-out compared to wild-type mice. These findings implicate an important apoD/AA interaction, which may be necessary for clozapine function.