A TLC procedure was developed to determine dihydropyrimidine dehydrogenase (DPD) activity in human peripheral lymphocytes. The assay, which used radiolabeled uracil as a substrate, was validated using recombinant pig DPD in which it was demonstrated to yield kinetic constants similar to those found by methods that rely on either spectroscopic determination of NADPH oxidation or HPLC. DPD activity was measured in a group of human lymphocyte extracts, including an extract from a subject that actually presented toxicity to 5-fluorouracil treatment. Measurements of DPD protein content using western immunoblots revealed a significant correlation with activity levels in human lymphocytes. Thus, this correlation could be used to determine not only the levels of expression of this enzyme, which is the cause of an inherited genetic deficiency in pyrimidine catabolism, but also to estimate the degree of sensitivity to pyrimidine-based cancer drugs such as 5-fluorouracil.