The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 184.108.40.206), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitrarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37 degrees C with a saturating concentration of exogenous thymidine. In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 +/- 11 units (mean +/- S.D., n = 20, range 45-89). The apparent Michaelis constant for thymidine was of the order of 10(-4) M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin. Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity +/- S.D. was 96 +/- 27 units; range 58-140, n = 8). Thymine formation from thymidine was temperature- and pH-dependent in whole blood. 2'-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3'-azido-3'-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously. In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.