Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growtharrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth . Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts . In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases . It was sensitive to Flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion . First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol -precipitable material) from endothelial cell-conditioned medium reconstituted in 20% serum inhibited smooth muscle cell growth ; glycosaminoglycans isolated from unconditioned medium (i .e ., 0.4% serum) had no effect on smooth muscle cell growth . No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase . Second, exogenous heparin, heparan sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20% serum and tested for their ability to inhibit smooth muscle cell growth . Heparin inhibited growth at concentrations as low as 10 ng/ml . Other glycosaminoglycans had no effect at doses up to 10 tug/ml . Anticoagulant and non-anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk . Nature (Lond.) . 265:625-626,1977; Guyton et al . Circ . Res. 46 :625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al . Circ. Res. 47 :578-583, 1980) . We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.A characteristic feature of the normal, healthy arterial wall is that the intimal endothelial cells form a continuous quiescent monolayer, and the underlying medial smooth muscle cells also remain in a quiescent growth state . If the endothelium is damaged, smooth muscle cell proliferation occurs until the endothelium regenerates (9, 29). The regulation of cell growth in the vascular wall is poorly understood . Ross (15, 28) and others (10,25) (11) have shown that endothelial cells produce a factor which stimulates the growth of smooth muscle cells. Eisenstein et al . (8) have found that extracts from the inner arterial wall can be fractionated to produce both stimulators and inhibitors of smooth muscle cell growth .We present evidence demonstrating that cultured endothelial cells produce both positive a...