Kupffer cells, endothelial cells, and hepatocytes were separated by centrifugal elutriation. The rate of uracil formation from [2-14C]uridine, the first step in uridine catabolism, was monitored in suspensions of the three different liver cell types. Kupffer cells demonstrated the highest rate of uridine phosphorolysis. 15 min after the addition of the nucleoside the label in uracil amounted to 51%, 13%, and 19% of total radioactivity in the medium of Kupffer cells, endothelial cells, and hepatocytes, respectively. If corrected for Kupffer cell contamination, hepatocyte suspensions demonstrated similar activities as endothelial cells. In contrast to non-parenchymal cells, hepatocytes continuously cleared uracil from the incubation medium. The lack of uracil consumption by Kupffer cells and endothelial cells points to uracil as the end-product of uridine catabolism in these cells. Kupffer cells and endothelial cells did not produce radioactive CO2 upon incubation in the presence of [2-14C]uridine. Hepatocytes, however, were able to degrade uridine into CO2, beta-alanine, and ammonia as demonstrated by active formation of volatile radioactivity from the labeled nucleoside. There was almost no detectable formation of thymine from thymidine or of cytosine, uracil, or uridine from cytidine by any of the different cell types tested. These results are in line with low thymidine phosphorolysis and cytidine deamination in rat liver. Our studies suggest a co-operation of Kupffer cells, endothelial cells, and hepatocytes in the breakdown of uridine from portal vein blood with uridine phosphorolysis predominantly occurring in Kupffer cells and with uracil catabolism restricted to parenchymal liver cells.