The involvement of collagen in cholesterol-induced atherosclerosis in rabbits was investigated. Rabbits were fed a 2% cholesterol diet for 8, 16, 30, 60 and 90 days. Histological sections were taken and aortic free and esterified cholesterol were determined after separation on thin-layer chromatography. Prolyl hydroxylase activity was used as a measure of collagen synthetic rate and hydroxyproline levels as an estimate of collagen content. Cholesterol content was significantly increased after 8 days, while at this time there were no gross aortic lesions. After 30 days there was some aortic disease and by 60 days most of the rabbits exhibited pronounced aortic lesions. Histologically, the lesions consisted mainly of intimal foam cells. There was no alteration in collagen synthetic rate or content at 8, 16, 30 or 60 days. These data indicate that 60 days of continuous cholesterol feeding results in a foam cell aortic lesion with no alteration in collagen metabolism. After 90 days of cholesterol feeding there was a significant increase in collagen synthetic activity in the thoracic aorta. These data suggest that alteration of collagen synthetic activity is a seconadry response, resulting from injury induced by the aortic accumulation of large amounts of cholesterol.