Studies using rat livers perfused with recycled, serum-containing medium plus [3H]leucine revealed that secreted VLDL contain three forms of apolipoprotein B (apoB), B-48, B-95, and B-100, all synthesized by the liver. The B-48/(B-95 + B-100) [3H]leucine incorporation ratio ranged from 0.22 to 3.25 with livers of rats fed different diets, and the ratio was positively correlated with the triglyceride secretion rate in most of the livers. Generally, as more triglyceride was secreted, a greater proportion was packaged with B-48, which is the apoB form most rapidly cleared from the circulation. Together, these findings suggest a mechanism for regulating plasma triglyceride levels. [3H]Leucine incorporation into apoA-I also was positively correlated with the triglyceride secretion rate. Secretion of newly synthesized B-48 was delayed relative to all other apolipoproteins. There was little segregation of any of the three apoB forms into any of five subfractions of secreted VLDL separated on the basis of Sf value; only the smallest VLDL (Sf 20-100) were slightly enriched in B-95 and B-100. Less than 5% of newly synthesized apoB appeared in perfusate LDL. The B-100/B-95 [3H]leucine incorporation ratio was 3.3 with perfused livers of fed rats but only 1.6 in post-surgical, relatively fasted rats in vivo, suggesting physiologic regulation also of the relative amounts of the two large apoBs produced. With recycled serum-free perfusate, as opposed to serum-containing medium, there was hepatic reuptake of nascent VLDL, indicated by the reuptake of newly synthesized apoE and all three forms of apoB, and not other apolipoproteins. Divergent metabolism of B-100 and B-95 in the rat was evident from the following results: a) B-95 disappeared more rapidly from recycled, serum-free liver perfusate; b) B-100 disappeared more rapidly from the circulation in vivo; c) plasma lipoprotein fractions of increasing density between d less than 1.019 and d 1.072 g/ml contained increasing proportions of B-95 over B-100. In summary, these results show that hepatic VLDL production in the rat involves the biosynthesis of three forms of apoB, that the relative amounts produced are regulated by physiologic variables, and that there is divergent metabolism of the VLDL particles into which these different apoB forms, either individually or in combination, become incorporated.