Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.