A scheme for the partial purification of human erythrocyte pyrimidine nucleotidase isozymes is presented. The characteristics of three of these were distinctively established. One isozyme, designated pyrimidine nucleotidase (PyrNase), is active for uridine monophosphate (UMP) and cytidine monophosphate (CMP), but not for thymidine monophosphate (dTMP). Two other isozymes, designated thymidine nucleotidase I (ThyNase I) and thymidine nucleotidase II (ThyNase II), were active for dTMP, but less so for UMP and CMP. Molecular weights of PyrNase, ThyNase I, and ThyNase II were estimated as 52,000, 52,000, and 48,000, respectively. Isoelectric points of these isozymes were 5.22, 4.90, and 4.68, respectively. Kinetic study revealed that the Km value for dTMP of ThyNase I was 3.83 mmol/L and of ThyNase II was 6.26 mmol/L.