Determination of circulating red cell volume (RCV) in anemic preterm infants is, in theory, a better indicator of transfusion needs than Hb concentration. Our study reports the results of RCV measurement using biotin labeling of red cells on 40 occasions in preterm infants of 25–34 wk gestation. In 20 infants, who had estimations made within 24 h of birth, the RCV varied between 17.7 and 66 mL/kg. Twenty measurements were made at a later age at the time of a blood transfusion. RCV values were between 13.1 and 41.5 mL/kg before transfusion. In 13 infants, RCV was determined simultaneously using two methods, biotin and dilution of autologous HbF with donor HbA at transfusion. There was no significant difference between the results of RCV estimations using these two methods. Our study demonstrates that biotin labeling is an effective method for determining RCV in preterm infants.