The failure of sheep red blood cells (RBCs) labeled with Chromium-51 (Cr-51) using the ascorbic acid technique to act as a suitable intravascular marker of blood volume in a septic sheep model prompted us to investigate the technique of radiolabeling sheep erythrocytes with this isotope. Consequently, we studied thirteen sheep in which the labeling efficiency of Cr-51 as sodium chromate and hemoglobin typing was determined for each animal. Mean Cr-51 labeling efficiency of sheep RBCs was 67.5% (n = 13). Although 5 of the 13 sheep were discovered to have two types of hemoglobin (Hb) as determined by electrophoresis, overall labeling efficiency of sheep RBCs was determined to be independent of the type of hemoglobin present. However, when two types of Hb were present (Hb-A and Hb-B), Cr-51 had a higher affinity for Hb-B (80%) than Hb-A (20%) even though both Hb types are present in similar proportions (Hb-A = 53%, Hb-B = 46%). The results of this study indicate that sheep RBCs express a lower labeling efficiency for Cr-51 than do human RBCs and that Cr-51 has a higher affinity for Hb-B than for Hb-A when both hemoglobin types are present. This difference is noteworthy when interpreting Cr-51 RBC data in experimental sheep models. Furthermore, caution should be exercised when extrapolating established human protocols to animal models.