We previously demonstrated that macrophages isolated from mice fed gamma-linolenic acid (GLA)-enriched diets reduce vascular smooth muscle cell (SMC) proliferation in a cyclooxygenase-dependent fashion and may therefore favorably modulate the atherogenic process. The present study was conducted to elucidate the mechanism(s) by which dietary GLA influences the ability of macrophages to modulate SMC growth programs. Resident peritoneal macrophages were isolated from C57BL/6 female mice fed diets containing variable GLA compositions at 10% (wt/wt), treated with various antibodies and co-cultured with cycling naive vascular SMC isolated from nonpurified diet-fed mice. Smooth muscle cell proliferation and intracellular cAMP levels were measured after co-culture. In parallel experiments, cycling naive vascular SMC isolated from nonpurified diet-fed mice were dosed with exogenous prostaglandin E1 (PGE1 ) for various periods and challenged with cycloheximide for 4 h (8-12 h after PGE1 addition), and intracellular cAMP levels were measured at various time points. Macrophages isolated from mice fed GLA-enriched dietary oils significantly reduced SMC proliferation in co-culture compared with controls (macrophages from mice fed a corn oil diet containing no GLA). Anti-PGE1 antiserum treatment (1:50 or 1:100) blocked the ability of GLA-enriched macrophages to down-regulate SMC proliferation, a response reversed by exogenous PGE1 treatment. Macrophages isolated from mice fed GLA-enriched dietary oils elevated SMC intracellular cAMP levels in a biphasic fashion. In addition, exogenous PGE1 (1 nmol/L to 10 micromol/L) exerted a similar biphasic cAMP response in SMC, and the second phase of cAMP elevation was antagonized by cycloheximide. In conclusion, dietary GLA enhances mouse macrophage-derived prostaglandin E1, which inhibits vascular SMC proliferation.