Erythrocyte 5'-nucleotidase is thought to be involved in the maturation of erythrocytes. In the present study, in vitro incubation of canine erythrocytes demonstrated that significant inhibition of 5'-nucleotidase activity occurred in the presence of serum from dogs infected with Babesia gibsoni, when the enzyme was assayed with cytidine 5'-monophosphate (5'-CMP) and inosine 5'-monophosphate (5'-IMP) as substrates. The multiplication of B. gibsoni in in vitro culture also resulted in a significant decrease in the enzyme activity of erythrocytes in the culture. Furthermore, the infected serum and 5'-CMP retarded the maturation of canine reticulocytes in vitro. These results suggested that nucleotides such as 5'-CMP and 5'-IMP might accumulate in young erythrocytes and/or serum in dogs infected with B. gibsoni as a result of decreased activity of erythrocyte 5'-nucleotidase, resulting in the delayed maturation of reticulocytes.