The ability of isolated nonparenchymal and parenchymal rat liver cells to metabolize 5beta-cholestane-3alpha,7alpha,12alpha-triol and other bile acid intermediates has been investigated. Incubation of nonparenchymal cells with 5beta-cholestane-3alpha,7alpha,12alpha-triol resulted in the formation of one more polar product identified as 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol. The formation was linear with time up to 2 hr and with the number of cells, and showed saturation kinetics with respect to substrate concentration. The maximum rate of conversion was 90 pmol/10(6) cells per hr. Incubation of hepatocytes with the triol resulted in the formation of several more polar products. In addition to 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol, products with retention time on high pressure liquid chromatography identical with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oic acid and cholic acid were observed. The identity of these products was verified by combined gas-liquid chromatography-mass spectrometry. The rate of conversion was linear with time for about 10 min and saturation with respect to substrate concentration was not attained. At identical substrate concentrations, the total rate of conversion (12.5 nmol/10(6) cells per hr) was at least two orders of magnitude higher than with the nonparenchymal cells. Similar differences in rates of conversion were observed with other C(27)-bile acid intermediates. It is concluded that the nonparenchymal cells do not play any significant role in the conversion of bile acid intermediates, either under physiological conditions or under experimental conditions where such steroids have been administered intravenously.-Dueland, S., J. I. Pedersen, C. A. Drevon, and I. Björkhem. 26-Hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol by isolated nonparenchymal cells and hepatocytes from rat liver.