—Endothelium-derived products have been implicated in the regulation of vascular wall structure through their effects on extracellular matrix metabolism. The purpose of this study was to further understand the paracrine mechanisms underlying endothelial cell regulation of extracellular matrix metabolism by testing the hypothesis that endothelium-derived nitric oxide decreases the concentration of soluble collagens derived from vascular smooth muscle cells (VSMCs). Porcine coronary endothelium and VSMCs were grown under a coculture configuration to assess the paracrine effects of nitric oxide produced by the endothelium on VSMC collagen types I and III. Endogenous endothelial cell nitric oxide production was blocked with N^ω-nitro-l-arginine methyl ester. Collagen type I and type III were quantitatively measured using an enzyme-linked immunosorbent assay method. The endothelium elicited a time-dependent increase in the concentration of soluble VSMC-derived collagen type I; in contrast, collagen type III was decreased. After inhibition of nitric oxide production, there was a marked increase in both collagen types I and III concentration. These results demonstrated that endothelium-derived nitric oxide differentially alters collagen subtypes produced by VSMCs. The data support the hypothesis that nitric oxide functions via a paracrine mechanism to decrease VSMC collagen types I and III concentration, a finding consistent with an integral role for the endothelium in modulating extracellular matrix metabolism.