The plasma and cellular pharmacology of 2',2'-difluorodeoxycytidine (dFdC, Gemcitabine) was studied during a phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC dose, which ranged between 53 and 1,000 mg/m2 per 30 min. The cellular pharmacokinetics of an active metabolite, dFdC 5'-triphosphate (dFdCTP), were determined in mononuclear cells of 22 patients by anion-exchange high-pressure liquid chromatography. The rate of dFdCTP accumulation and the peak cellular concentration were highest at a dose rate of 350 mg/m2 per 30 min, during which steady-state dFdC levels of 15-20 microM were achieved in plasma. A comparison of patients infused with 800 mg/m2 over 60 min with those receiving the same dose over 30 min demonstrated that the dFdC steady-state concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by mononuclear cells incubated in vitro was maximal at 10-15 microM dFdC. These studies suggest that the ability of mononuclear cells to use dFdC for triphosphate formation is saturable. In the design of future protocols, a dose rate should be considered that produces maximal nucleotide analogue formation, with increased intensity being achieved by prolonging the duration of infusion.