Glycerolipid and apoB synthesis and secretion were examined in hepatocytes obtained from fetal, suckling (day 6), and adult rats in order to examine the developmental regulation of lipoprotein production. The capacity to synthesize [3H]triacylglycerol (from [3H]glycerol) followed the order: adult greater than day 6 greater than fetal. Addition of 1 mM oleic acid to the incubation media stimulated the incorporation of [3H]glycerol into triacylglycerol 6.7- and 3.6-fold by fetal and adult hepatocytes, respectively. After maximal stimulation by 1 mM oleic acid, triacylglycerol secretion by fetal cells was still only 39% of the amount secreted by adult cells that had been treated similarly. Fetal cells stimulated with 1 mM oleic acid synthesized the same amount of triacylglycerol as adult cells that had been treated with 0.1 mM oleic acid. However, the fetal cells secreted only one-third as much triacylglycerol, further demonstrating relatively impaired secretion of triacylglycerol. In order to determine whether low triacylglycerol secretion was associated with differences in apoB metabolism, cells were incubated with [35S]methionine and apoB was quantified after immunoprecipitation. Fetal cells synthesized and secreted nearly equal amounts of large molecular weight and small molecular weight apoB. In contrast, adult cells synthesized and secreted nearly twice as much small molecular weight apoB as large molecular weight apoB. Moreover, although fetal and adult cells secreted large molecular weight apoB at similar rates, adult cells synthesized and secreted small molecular weight apoB at rates that were nearly two times higher than fetal cells. These data suggest that the ability to assemble and secrete VLDL varies in parallel with the developmental expression of small molecular weight apoB. These studies also show the usefulness of the cultured rat hepatocyte model for examining the ontogeny and regulation of lipoprotein assembly/secretion.