This study was conducted to compare the impact of dietary lipids on the ability of macrophages to modulate vascular smooth muscle cell (SMC) DNA synthesis in vitro. C57BL/6 female mice were fed six different diets (6 mice/diet) containing 10% fat from corn oil (CO), borage oil (BO), primrose oil (PO), fish-corn oil mix (FC, 9:1, w/w), fish-borage oil mix (FB, 1:3, w/w), or fish-primrose oil mix (FP, 1:3, w/w) for 2 wk. Peritoneal macrophages were isolated from these mice, stimulated with zymosan or vehicle, and subsequently co-cultured with naive mouse aortic SMC in the presence of 3H-thymidine to measure SMC DNA synthesis. In this co-culture system, macrophages were seeded on 25-mm culture inserts (upper chamber) and SMC were seeded on 35-mm culture dishes (lower chamber). The two cell types were separated by a semipermeable membrane with a 30-kD cut-off. When quiescent SMC were co-cultured with macrophages, only the PO and FP diet groups had significantly (P < 0.05) lower SMC DNA synthesis compared with the control CO group whose diet contained no gamma-linolenic acid (GLA) or (n-3) polyunsaturated fatty acids (PUFA). In contrast, when cycling SMC were co-cultured with diet-modulated macrophages, all dietary groups except for those fed FC had significantly lower (P < 0.05) SMC DNA synthesis relative to the CO group. Although the level of GLA in PO and BO diets was different (11.5 and 22.3 g/100 g fatty acids, respectively), these treatments exerted comparable inhibitory effects on SMC DNA synthesis. The FP treatment consistently exhibited the lowest SMC DNA synthetic profile among the six dietary groups irrespective of SMC growth conditions. These data suggest that BO and PO alone or in combination with fish oil influence macrophage/smooth muscle cell interactions in a manner consistent with favorable modulation of the atherogenic process.