—Recent studies indicate that vascular smooth muscle cells (VSMCs) generate CO from the degradation of heme by the enzyme heme oxygenase-1 (HO-1). Because platelet-derived growth factor (PDGF) modulates various responses of VSMCs, we examined whether this peptide regulates the expression of HO-1 and the production of CO by rat aortic SMCs. Treatment of SMCs with PDGF resulted in a time- and concentration-dependent increase in the levels of HO-1 mRNA and protein. Both actinomycin D and cycloheximide blocked PDGF-stimulated HO-1 mRNA and protein. In addition, PDGF stimulated the production of reactive oxygen species by SMCs. Both the PDGF-mediated generation of reactive oxygen species and the induction of HO-1 protein was inhibited by the antioxidant N-acetyl-l-cysteine. Incubation of platelets with PDGF-treated SMCs resulted in a significant increase in platelet cGMP concentration that was reversed by treatment of SMCs with the HO-1 inhibitor tin protoporphyrin-IX or by addition of the CO scavenger hemoglobin to platelets. In contrast, the nitric oxide inhibitor methyl-l-arginine did not block the stimulatory effect of PDGF-treated SMCs on platelet cGMP. Finally, incubation of SMCs with the releasate from collagen-activated platelets induced HO-1 protein expression that was blocked by a neutralizing antibody to PDGF. These results demonstrate that either administered exogenously or released by platelets, PDGF stimulates HO-1 gene expression and CO synthesis in vascular smooth muscle. The ability of PDGF to induce HO-1–catalyzed CO release by VSMCs may represent a novel mechanism by which this growth factor regulates vascular cell and platelet function.