Thrombospondin (TS) is an extracellular glycoprotein whose synthesis and secretion by vascular smooth muscle cells (SMC) is regulated by platelet-derived growth factor. We have used a panel of five monoclonal antibodies against TS to determine an essential role for thrombospondin in the proliferation of cultured rat aortic SMC. All five monoclonal antibodies inhibited SMC growth in 3-d and extended cell number assays; the growth inhibition was specific for anti-TS IgG. The effects of one antibody (D4.6) were examined in detail and were found to be reversable and dose dependent. Cells treated with D4.6 at 50 micrograms/ml (which resulted in a greater than 60% reduction in cell number at day 8) were morphologically identical to control cells. D4.6-treated SMC were analyzed by flow cytofluorimetry and were found to be arrested in the G1 phase of the cell cycle. To determine a possible cellular site of action of TS in cell growth, SMC were examined by immunofluorescence using a polyclonal antibody against TS. TS was observed diffusely bound to the cell surface of serum- or platelet-derived growth factor-treated cells. The binding of TS to SMC was abolished in the presence of heparin, which prevents the binding of TS to cell surfaces and inhibits the growth of SMC. Monoclonal antibody D4.6, like heparin, largely abolished cell surface staining of TS but had no detectable effect on the cellular distribution of fibronectin. These results were corroborated by metabolic labeling experiments. We conclude that cell surface-associated TS is functionally essential for the proliferation of vascular SMC, and that this requirement is temporally located in the G1 phase of the cell cycle. Agents that perturb the interaction of TS with the SMC surface, such as heparin, may inhibit SMC proliferation in this manner.