The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.