Vitreous from bovine, human and chick embryo has been found to contain a trypsin inhibitory activity. Chymotrypsin-inhibitory activity was also identified in bovine and chick embryo vitreous. Following either ultrafiltration or Bio Gel P-10 chromatography, these activities appear in fractions having a molecular weight greater than 10000 MW (ultrafiltration) or greater than 13000 MW (P-10 void volume), and are separable from low molecular weight aortic endothelial cell growth inhibitory activity present either in the ultrafiltrate or P-10 retarded volume. Treatment of the trypsin inhibitory fraction with hyaluronidase had no effect on trypsin inhibition, nor did addition of hyaluronic acid inhibit trypsin. Chick embryo vitreous and hyalocyte-conditioned medium were found to contain aortic endothelial cell growth inhibitory activity in both the void volume and retarded volume fractions following Bio Gel P-10 chromatography. Both the 6200 MW bovine vitreous endothelial cell growth inhibitor and the high molecular weight chick embryo vitreous endothelial cell growth inhibitor (greater than 13000 MW) were similar, in that most of the activity did not bind to heparin linked to Sepharose CL-6B.