Proinflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders. We have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) induces a catalytic activation of the serotonin transporter (SERT) arising from a reduction in the SERT Km for 5-hydroxytryptamine (5-HT). As inflammatory cytokines can activate p38 MAPK, we hypothesized that they might also activate neuronal SERT. Indeed, Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulated serotonin uptake in both the rat embryonic raphe cell line, RN46A, and in mouse midbrain and striatal synaptosomes. In RN46A cells, IL-1beta stimulated 5-HT uptake in a dose- and time-dependent manner, peaking in 20 min at 100 ng/ml. This was abolished by IL-1ra (20 ng/ml), an antagonist of the IL-1 receptor, and by SB203580 (5 microM), a p38 MAPK inhibitor. TNF-alpha also dose- and time-dependently stimulated 5-HT uptake that was only partially blocked by SB203580. Western blots showed that IL-1beta and TNF-alpha activated p38 MAPK, in an SB203580-sensitive manner. IL-1beta induced an SB203580-sensitive decrease in 5-HT Km with no significant change in Vmax. In contrast, TNF-alpha stimulation decreased 5-HT Km and increased SERT Vmax. SB203580 selectively blocked the TNF-alpha-induced change in SERT Km. In mouse midbrain and striatal synaptosomes, maximal stimulatory effects on 5-HT uptake occurred at lower concentrations (IL-1beta, 10 ng/ml; TNF-alpha, 20 ng/ml), and over shorter incubation times (10 min). As with RN46A cells, the effects of IL-1beta and TNF-alpha were completely (IL-1beta) or partially (TNF-alpha) blocked by SB203580. These results provide the first evidence that proinflammatory cytokines can acutely regulate neuronal SERT activity via p38 MAPK-linked pathways.