Dopaminergic function is thought to be altered in major depression and, in animal studies, is reduced in omega-3 polyunsaturated fatty acid (PUFA) deficiency states. Therefore we studied PUFAs and resting prolactin, a marker for dopaminergic tone, and cerebrospinal fluid homovanillic acid (HVA), the chief dopamine metabolite. In medication-free adults (n = 23) with DSM-IV major depressive disorder (MDD), we measured plasma phospholipid levels of omega-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the omega-6 PUFA arachidonic acid (AA), and plasma prolactin levels before and after administration of dl-fenfluramine (FEN). In a subset of patients (n = 14), cerebrospinal fluid levels of HVA and the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were obtained through lumbar puncture. Baseline prolactin was negatively correlated with omega-3 PUFAs (logDHA, F
1,21 = 20.380, p < 0.001; logEPA, F
1,21 = 10.051, p = 0.005) and positively correlated with logAA:DHA (F
1,21 = 15.263, p = 0.001), a measure of omega-6/omega-3 balance. LogDHA was negatively correlated with CSF HVA (Spearman's ρ = −0.675, p = 0.008) but not 5-HIAA (Spearman's ρ = −0.143, p = 0.626) after controlling for sex and HVA – 5-HIAA correlation. PUFAs did not predict the magnitude of the FEN-stimulated change in prolactin, considered to be a serotonin effect. The robust relationship of omega-3 PUFAs with dopaminergic but not serotonergic indices suggests that omega-6:omega-3 balance may impact depression pathophysiology through effects on the dopaminergic system.