Background: The incidence of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in Iran is estimated at 10-14.9%. The donor blood in blood banks is not screened routinely for this enzyme deficiency and such blood may be used for neonatal exchange transfusion.Objective: To study the effect of G-6-PD deficient blood in neonatal exchange blood transfusion.Methods: In a prospective study, serum bilirubin was checked before and 6 hours after exchange transfusion in three hundred and fifty consecutive neonates who were admitted to Nemazee Hospital, Neonatal Ward. Hemoglobin, direct Coomb’s test, direct bilirubin, reticulocyte count, blood group of neonates and mothers, G-6-PD of neonates and the blood used for exchange transfusion were also checked. For analysis of the data, 102 neonates who weighed more than 2500 gr and with no evidence of hemolysis, liver disease or sepsis were divided into three groups; Group I: patients in this group were exchanged with G-6-PD deficient blood. Group IIa: patients in this group were exchanged with normal blood. Group IIb: the neonates from Group IIa who needed a repeat exchange transfusion. Group III: G-6-PD deficient patients who were exchanged with normal blood. Results: Group I neonates had a small drop in bilirubin 6 hours post-exchange transfusion in contrast to groups II and III (P<0.05). Exchange transfusion with G-6-PD deficient donor blood led to repeat exchange transfusion due to insufficient fall in bilirubin level in Group I neonates.Conclusion: It is recommended that in areas endemic for G-6-PD deficiency, the donor blood be screened before exchange transfusion.