The mechanism of erythrocyte senescence remains an unsolved problem. Even though the study of genetically determined hemolytic anemias has demonstrated a variety of defects which can shorten the erythrocyte life span, no clear cut answer has emerged as to the normal mechanisms which determine erythrocyte survival. The examination of erythrocyte senescence has been hindered by the difficulty of isolating aged cells. A variety of techniques have been utilized for obtaining these senescent erythrocytes but most are not satisfactory. This proposal describes a new procedure for the isolation of senescent erythrocytes from the rabbit. With this technique, rabbit erythrocytes are covalently labeled with biotin by reaction with N-hydroxysuccinimido biotin. The bound biotin has been shown to not affect erythrocyte survival when the derivatized cells are infused into the rabbit. At various times after infusion, the biotinylated erythrocytes can be selectively recovered by avidin affinity chromatography. With this procedure, erythrocytes are routinely isolated within 5 to 10 days of their expected death. These senescent erythrocytes have been found to have normal levels of adenosine 5' - triphosphate. The present proposal will examine additional characteristics of these senescent erythrocytes including the integrity of the glycolytic and hexosemonophosphate pathways, the enzymes related to adenine nucleotide metabolism, the state of the cellular anti-oxidant defenses, the level of antibodies on the cell surface and the integrity of the skeleton/membrane complex. A careful examination of these parameters should lead to a better understanding of factors which control the normal senescence of erythrocytes.