Combinations of angiostatic steroids and heparin induce basement membrane breakdown, capillary retraction, and endothelial cell rounding as part of their antiangiogenic action (Ingber DE, Madri JA, Folkman J: Endocrinology 119: 1768, 1986). Specific modulators of matrix metabolism were examined for their effects and on capillary development in the growing chick chorioallantoic membrane to explore the possibility that structural alterations of extracellular matrix (ECM) could be causally involved in the antiangiogenic mechanism. Regression of growing capillaries was induced by proline analogs (1-azetidine-2-carboxylic acid, cis-hydroxyproline, d, L-3, 4-dehydroproline, thioproline) and an inhibitor of prolyl hydroxylase (alpha, alpha-dipyridyl) which interfere with triple helix formation and prevent collagen deposition. Beta-aminopropionitrile, an inhibitor of collagen cross-linking, was also antiangiogenic although an inhibitor of glycosaminoglycan deposition (beta-methyl d-xyloside) was not. Combination of suboptimal doses of active collagen modulators with either angiostatic steroids or heparin resulted in great potentiation of their antiangiogenic effects. Coadministration of proline analogs, angiostatic steroids, and heparin resulted in complete inhibition of angiogenesis (avascular zones in 100% of chorioallantoic membranes) and larger and more extensive avascular zones than previously observed with steroid-heparin combinations. The antiangiogenic effects of these compounds (alone and in combination) were found to be directly related to their ability to inhibit collagen accumulation. Screening of additional compounds that are known to alter ECM turnover resulted in identification of a new angiogenesis inhibitor, all trans-retinoic acid. These findings suggest that growing capillaries may be" switched" into a regressive mode through alterations of collagen metabolism that result in loss of ECM structural integrity. Identification of ECM turnover as a major control point in angiogenesis may also provide a more rational approach for development of new antiangiogenic regimens.