We examined the high affinity binding, uptake, and degradation of apo E-free 125I-high density lipoprotein (HDL) in cultured pig hepatocytes. At steady state, the cells degraded 9.4% of cell-associated 125I-HDL/hour, compared with 41.7%/hour for 125I-LDL. Pulse-chase experiments at 4 degrees C revealed that high affinity 125I-HDL binding was reversible. Similar experiments at 37 degrees C revealed that about 70% of the cell-associated 125I-HDL was released as a macromolecule; the remainder was degraded to acid-soluble products. In contrast, over 75% of the 125I-LDL that was released had been degraded to acid soluble products. The amount of macromolecular 125I-HDL released at 37 degrees C was similar to the amount that was bound to the cell surface, as estimated from measurements of trypsin-releasable radioactivity. Density gradient ultracentrifugation and SDS-polyacrylamide gel electrophoretic analysis of macromolecular 125I-HDL released to the medium revealed an increase in density, and the apparent partial proteolysis of apo A-I (Mr 25,000) to products of apparent Mr 12,000-14,000. The findings suggest that high affinity 125I-HDL uptake had a reversible component in which HDL was concentrated temporarily at the cell surface, modified, and then released as a somewhat denser lipoprotein particle. Measurement of 125I-HDL and 125I-LDL degradation in cell homogenates revealed no difference in the inherent susceptibility of the two lipoproteins to proteolysis by lysosomal enzymes. The overall slower rate of degradation of 125I-HDL compared to 125I-LDL was therefore due in part to the smaller fraction of HDL that was committed to irreversible catabolism. The rate of catabolism of this fraction, however, was considerable. Cells pulsed at 4 degrees C and subsequently warmed to 37 degrees C released one-half the acid-soluble products from 125I-HDL within about 4 hours, compared with 2 hours for cells pulsed with 125I-LDL. These findings indicate that HDL was internalized, transported to lysosomes, and degraded at about one-half the rate of LDL.