Smooth muscle cell (SMC) proliferation is a poorly understood process that plays a critical role in several pathological states, including atherosclerosis and hypertension. Recent work suggests that the oncogene c-myb and myosin, a ubiquitous cytoskeletal protein, may be directly involved in this process. We have used antisense nonmuscle myosin heavy chain (NMMHC) or c-myb phosphorothiolate oligonucleotides to inhibit proliferation of SMCs in vitro. The suppression of growth is accompanied by reductions in the concentrations of NMMHC and c-myb mRNAs as well as decreases in the levels of the corresponding proteins. The specificity of the antiproliferative effect is underscored by the absence of any detectable growth inhibition with sense NMMHC or c-myb phosphorothiolate oligonucleotides, an antisense c-myb mismatch phosphorothiolate oligonucleotide, or an antisense thrombomodulin phosphorothiolate oligonucleotide. Furthermore, the treatment of SMCs with antisense phosphorothiolate oligonucleotides for as little as 2 hours causes maximal inhibition of cell growth over the next 72 hours. Under these conditions, SMCs attain normal rates of growth over the following 48 hours, which shows that proliferation is suppressed in a reversible fashion by antisense phosphorothiolate oligonucleotides. These experiments indicate that both c-myb and nonmuscle myosin play critical roles in SMC proliferation and that reductions of either mRNA by antisense phosphorothiolate oligonucleotides arrest the process.