Abstract: The human melanoma cell line A2058 expresses the G_q‐coupled M5 subtype of muscarinic receptor. Stimulation with the cholinergic agonist, carbachol, induces a dose‐dependent increase in arachidonic acid release. The carbachol‐induced arachidonate release is potentiated two‐ to threefold by pretreatment of A2058 cells with either of the inflammatory cytokines, tumor necrosis factor‐α or interleukin‐1β. Cytokine‐induced enhancement of muscarinic‐mediated arachidonic acid release peaks near 1 h. Western analysis suggests that both cytokines are capable of activating the nuclear factor‐κB (NF‐κB) and p38 mitogen‐activated protein kinase (MAPK) pathways. Anisomycin (1 μM) treatment mimics the cytokine‐induced enhancement of arachidonic acid production and activates the p38 MAPK pathway, but does not activate the NF‐κB pathway. Furthermore, pretreatment of A2058 cells with the putative p38 MAPK inhibitor, SB202190, ablates the cytokine‐dependent augmentation without interfering with the muscarinic‐mediated arachidonic acid release in untreated cells. Moreover, cytokine treatment does not affect other M5‐coupled pathways (e.g., phospholipase C activity or intracellular Ca²⁺ mobilization), suggesting that p38 MAPK activation principally modulates muscarinic‐mediated phospholipase A₂ activity. Finally, in primary cultures of cells taken from rat cerebellum, key aspects of this finding are repeated in cultures enriched for glia, but not in cultures enriched for granule neurons.