Electrophoretic and quantitative analysis of pyrimidine 5'-nucleotidase in human erythrocytes from healthy individuals and a patient deficient in pyrimidine 5'-nucleotidase, using a range of substrates, has shown that the patient has a marked deficiency with UMP, CMP and dCMP as substrates but near normal levels of activity with dUMP and dTMP as substrates. The observations suggest that two separate structural gene loci coding for distinct 5'-nucleotidases with similar electrophoretic mobility exist in man. The genetic determination of these enzymes seems therefore to be homologous with that found in rodents.