The sheep is a useful model to study fetal and newborn physiology including perinatal erythropoiesis and red cell kinetics. A practical, economical method for measuring red cell survival (RCS) in sheep would be very valuable. However,51Cr is unsatisfactory, and suitable alternatives have not been published. In the course of investigating [14C]cyanate as a label for sheep red cells, we observed continued covalent labeling over 24 hin vivothat was great enough to introduce a substantial artifact into two commonly used parameters of RCS: posttransfusion recovery(PTR24) and time to 50% decrease (T50) when referenced to time zero. In a simulation ofin vivoconditions, the amount of14C bound to Hb increased 26 ± 6% (mean ± 1 SD,n= 11) over 24 h. To investigate the mechanism of the increasing14C bound, acid-acetone extraction, molecular sieve chromatography, and density gradient separation were used separately or in combination to quantitate intracellular free14C and14C covalently bound to intracellular proteins. Free14C decreased as protein-bound[14C]cyanate increased. These studies provide evidence that covalent binding of [14C]cyanate to intracellular Hb continuesin vivofor the first 24 h and that the source of the increase is intracellular free[14C]cyanate. We conclude that1) PTR24cannot be accurately determined by [14C]cyanate unless labeled red cells are incubated before infusion to allow the cyanate reaction to approach completion and2) RCS by [14C]cyanate should be referenced to blood concentrations at 24 h.Abbreviations: RCV,red cell volume;RCS,red cell survival;PTR24,posttransfusion recovery at 24 h;T50,time to decline 50% from reference value