Partial and reversible impairment of bile formation has been reported to occur in the offspring of rats undergoing common bile duct ligation during the last third of pregnancy. This situation was defined as latent cholestasis of the neonate and was suggested to be related to the multilamellar bodies partially occupying the canalicular lumen. The current study was undertaken to investigate the presence of alterations in the secretion of biliary lipids in these infant rats. Using both high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses, no changes caused by maternal cholestasis were found in either the conjugation pattern, or in the ratio of primary to secondary major bile acids in bile samples collected from 4-week-old and 8-week-old rats. However, a decrease in the proportion of cholate together with an increase in the amount of α- and omega-muricholate were found at 4 weeks of age. These changes were different from those observed in the pattern of maternal plasma bile acids, in which β-, but not α-muricholate, concentrations were increased. Moreover, studies performed by labeling the bile acid pool of the cholestatic mother-fetus tandem with [14C]glycocholic acid (GC) at day 16 of pregnancy indicated that only a minor proportion (approximately 10%) of bile acids found in 4-week-old pups was of maternal origin. Changes in the bile acid pool composition were fully reversed by 8 weeks of age. Bile lecithin and cholesterol output were determined by enzymatic techniques, both under basal conditions and during stepwise taurocholate (TC) infusion. At the time when multilamellar bodies were found, i.e., 4 weeks after birth, no change in either nonstimulated or TC-induced cholesterol output was observed. By contrast, both spontaneous and TC-induced lecithin secretion were markedly higher (+200%) in pups of cholestatic mothers as compared with control rats. These differences were abolished at 8 weeks of age. At this time, cholesterol output was significantly lower than that found in younger animals. This reduction was more pronounced in the control than in the cholestatic group. Histological examination of liver samples collected from the cholestatic group at 4 weeks of age revealed the presence of multilamellar bodies not only in the canalicular lumen but also within vesicular structures located in the pericanalicular area or near the Golgi apparatus. Both intracellular and intracanalicular bodies were present before and after TC infusion for 2 hours. These results indicate that maternal cholestasis in rats induces profound alterations in biliary lipids and bile acid secretion in their pups. Because bile acids are important activators of different steps responsible for biliary lipid secretion (intracellular trafficking, releasing into bile, and solubilization), alterations in maternal bile acid pool size and composition may affect the fetal development of biliary lipid secretion mechanisms, which may result in the appearance of multilamellar bodies within bile canaliculi, which in turn may be involved in the reversible latent cholestasis observed in these infants rats.