Heparin inhibits arterial smooth muscle cell (SMC) proliferation in vivo and in vitro; moreover, it reinduces the expression of alpha-smooth muscle (SM) actin (an accepted marker of SMC differentiation) in SMCs of the intimal thickening that develops after experimentally induced endothelial lesions. We have investigated the effect of heparin on the proliferation and actin isoform expression in cultured rat SMCs. In the presence of 10% fetal calf serum (FCS), heparin-treated primary and passage 5 SMCs showed a decrease of proliferation and an increase of alpha-SM actin (measured by Western blots or two-dimensional gel electrophoresis) compared with untreated SMCs. When SMCs were cultured in the presence of 10% plasma-derived serum, no proliferation occurred and heparin did not modify alpha-SM actin expression. This suggests that the action of heparin is related to its antiproliferative activity. SMCs cultured in the presence of 10% FCS plus heparin had the same level of proliferation as SMCs cultured in 5% FCS but had a higher content of alpha-SM actin. SMCs cultured in 20% rat whole-blood serum had a proliferation similar to that observed in SMCs cultured in 10% FCS but had a higher content of alpha-SM actin. Moreover, in SMCs cultured in 20% whole-blood serum, heparin inhibited SMC proliferation but did not modify alpha-SM actin expression. Thus, the action of heparin on alpha-SM actin expression appears to be partially independent of proliferation and is related to culture conditions. The proportion of alpha-SM actin mRNA, as measured by Northern blots with an alpha-SM actin mRNA-specific probe, was increased by heparin compared with cells cultured in 10% FCS; this suggests that heparin acts at the transcriptional or posttranscriptional level. Our results show that heparin acts not only on SMC proliferation but also on SMC differentiation; further investigation along these lines may help in the understanding of the mechanisms of SMC adaptation during normal and pathological conditions.