The mode of permeation of uracil, 5-fluorouracil, and orotic acid into cells has been investigated in four established cell lines (Novikoff rat hepatopma, P388 mouse leukemia, mouse L., and Chinese hamster ovary cells) in attempts to assess the rate-determining step(s) in their incorporation into the nucleotide pool and nucleic acids. Uracil and 5-fluorouracil shared a saturable transport system (Km = 5 to 15 mM) capable of rapid equilibration of these substrates across the cell membrane (t 1/2 at 25 degrees in first-order range of concentration = 25 to 58 sec). Thus it seems unlikely that transport is limiting the incorporation of uracil or fluorouracil. Their transport was inhibited by various nucleosides and hypoxanthine. Only the non-ionized form of fluorouracil was a substrate for the transporter; exclusion of charged pyrimidines may explain why orotate was not a substrate at physiological pH. Orotate permeated the cell membrane much more slowly (t 1/2 = 2890 to 6930 sec); its permeation was apparently non-mediated and rate-determining in the conversion of extracellular orotate to intracellular nucleotides.