The aim of this study was to determine the effect of ethanol on vascular smooth muscle cell proliferation and mitogen activated protein kinase (MAPK) signaling. Rat aortic smooth muscle cell growth in vitro was determined by measuring cell counts and [3H]thymidine incorporation. MAPK signaling was determined by assessing MEK (also referred to as MAPK kinase) activity by measuring phosphorylated extracellular signal-regulated kinase (pp44ERK - 1 and pp42ERK - 2) expression, and ERK activity by measuring ERK-2-dependent phosphorylation of myelin basic protein (MBP). In quiesced smooth muscle cells, ethanol treatment (24 h) inhibited serum-stimulated mitogenesis in a dose-dependent manner, (IC50 = 60 mM), in the absence of any effect on smooth muscle cell viability. In addition, ethanol treatment caused a significant shift to the right in the smooth muscle cell growth curve, extending the population doubling time from approximately 48 h (control) to approximately 70 h (ethanol). Acute (15 min) ethanol treatment reduced serum-stimulated pp44ERK - 1 and pp42ERK - 2 expression in a dose dependent fashion; 24.5+/-1.5% and 77.6+/-3.2% inhibition for 20 mM and 160 mM ethanol, respectively. Furthermore, there was a significant dose-dependent decrease in ERK2 activity in ethanol treated smooth muscle cells as compared to control smooth muscle cells. These data demonstrate an inhibitory effect of ethanol on smooth muscle cell proliferation and MAPK signalling in vitro. It is tempting to speculate that these actions of ethanol may contribute to its cardiovascular effects in vivo.