A flow-through system was used to study the cellular pharmacokinetics of 5-fluorouracil (5-FU) in four human cell lines (squamous-cell carcinoma HEp-2, colon carcinoma WiDr, hepatoma Hep G2, and breast carcinoma MCF-7) as well as in the rat hepatoma H35 cell line and in freshly isolated rat hepatocytes. The system made it possible to restrict the decrease in the concentration of 5-FU in the medium, to keep the volume in which the metabolites accumulated relatively small, and to study the dynamics of a response during and after a change in the composition of the eluent. Clearance of 5-FU from the eluent was achieved predominantly (greater than 95%) by its catabolism to dihydrofluorouracil in the tumor cell lines and to 2-fluoro-beta-alanine in the hepatocytes. Not only rat hepatocytes but also HEp-2 cells showed relatively high clearance values. A concentration-dependent 5-FU elimination was observed, indicating saturation of 5-FU elimination according to Michaelis-Menten kinetics (Km 14-22 microM). The maximal velocity (Vmax) values ranged from 0.025 to 0.13 nmol 5-FU/10(6) cells per minute. For HEp-2 cells, high-concentration pulse injections of 5-FU, thymine, uridine, or uracil immediately led to a reduction in 5-FU conversion, followed by recovery within 5 min. The flow-through system proved to be adequate for the study of the non-linear pharmacokinetics of 5-FU in different intact cells and for the comparison of various manipulations of these pharmacokinetics.