In two subjects the specific activity of esterified cholesterol in plasma lipoprotein subfractions was measured for up to 9 hr after an intravenous injection of [(3)H]mevalonic acid. It was found to be consistently higher in larger (S(f) > 100) than in smaller (S(f) 20-100) very low density lipoproteins (VLDL). Four subjects were given an intravenous injection of heparin so that the VLDL could be studied as its concentration fell and subsequently rose again. During the first hour the relative reduction was greatest for triglyceride, intermediate for free cholesterol, and least for esterified cholesterol. Between 1 and 7 hr postheparin, the VLDL pool was restored, but the pattern of increase of individual lipids was not parallel. The triglyceride increment was much greater during the 1-4-hr period than during the 4-7-hr period; in three of the subjects the free cholesterol increment was also greater during the earlier period. The increase in esterified cholesterol, however, was consistently greater during the 4-7-hr period. In six other subjects the specific activity of VLDL esterified cholesterol was related to that of its possible plasma precursors in samples collected at 1-hr intervals for 8 hr after the injection of [(3)H]mevalonic acid. Free cholesterol emerged as the most likely immediate precursor with the possibility of a hepatic as well as an intraplasma origin. The results did not support a major in vivo transfer of esterified cholesterol from high density lipoproteins to VLDL.