Mature human red cell metabolism was studied in an incubation system in which pH, pO 2, metabolic end-product, glucose, and co-factor substances could be maintained at predetermined concentrations in the red cell suspension for extended periods of time. The apparatus described, which could be developed for automated operation, utilized a membrane which allowed continual replacement of buffer without loss of cells from the incubation chamber, and complete collection of metabolic end-products. It was shown that the high sensitivity of glycolytic enzyme-substrate reactions to pH change, and the rapid response of glycolysis rate to ATP energy demands produced by changing incubation environment, required such systems if advances in standardization of measurement of metabolic rates in vitro were to be made. Continual buffer replacement proved a highly effective means of exercising a high degree of control in vitro over the concentration of rate-limiting co-factors, such as adenine, in the suspension medium. Observations could be made at optimal levels of metabolic function, and under varying degrees of cell adenine nucleotide availability while the same cells served as their own experimental controls. Comparisons of total endowment of enzymatic capacity as measured by the relative ability of cells from different individuals to process substrate, it was postulated, required that the naturally occurring variation in adenine nucleotide content of red cells be taken into account. More rapid equilibration of specific activity while cells were maintained at sustained desired levels of metabolic function, afforded the opportunity for highly advantageous applications of radiotracers to kinetic and radioisotope dilution studies.