Although circulating red blood cell (RBC) volume is a better measure of total body oxygen delivering capacity than hematocrit (HCT), circulating RBC volume is more difficult to measure. Thus, the HCT is often used in RBC transfusion decisions. However, several previous studies of low birth weight infants have reported that the correlation between HCT and circulating RBC volume is poor. Using a robust nonradioactive method based on in vivo dilution of biotinylated RBC enumerated by flow cytometry, the present study reexamined the correlation between HCT and circulating RBC volume in very low birth weight infants. Venous and capillary HCT levels were compared with circulating RBC volume measured using the biotin method. Twenty-six stable very low birth weight infants with birth weights less than 1300 g were studied on 43 occasions between 7 and 79 d of life. Venous HCT values correlated highly with circulating RBC volume (r ϭ 0.907; p Ͻ 0.0001). However, the mean 95% confidence limits for prediction of circulating RBC volume from venous HCT (the average error of prediction) was Ϯ13.4 mL/kg. The correlation between HCT and circulating RBC volume is strong in older stable very low birth weight infants. However, clinically important uncertainty exists in estimating circulating RBC volume and the associated RBC transfusion needs of an individual infant based on venous HCT. Intensive care of critically ill newborn infants has increased blood sampling and exacerbated the severity of neonatal anemia. Daily phlebotomy blood losses of 4 to 5% of the total blood volume are common among VLBW infants (1-3) who are defined here as those infants with birth weight less than 1500 g. Not surprisingly, the majority of the multiple RBC transfusions such infants receive are administered during the first weeks of life when phlebotomy loss is greatest (1, 2, 4, 5). The present consensus is that phlebotomy loss from clinical monitoring is the primary cause of the relatively large transfusion needs of VLBW infants (1, 3, 6, 7).The decision to administer a RBC transfusion is typically made on the basis of the infant's clinical condition and the whole blood HCT or blood Hb concentration (3,6,8). Although circulating RBC volume is a more accurate indicator of total body oxygen delivery capacity than whole blood HCT or Hb concentration (9, 10), HCT and Hb concentration are used clinically because they are quick, inexpensive, and analytically reliable. To understand the limitations of HCT as a guide to transfusion decisions, the relationship between HCT and circulating RBC volume in VLBW infants should be elucidated, and the sources of the previously reported poor correlation (9 -14) should be segregated into methodologic artifacts and genuine characteristics of preterm physiology.The aim of the present study was to examine the relationship between HCT and circulating RBC volume in stable VLBW infants. An accurate nonradioactive method based on biotinylated RBC was used to measure circulating RBC volume. ABSTRACT 525