The distribution of hexokinase isoenzymes was analyzed in erythrocytes from adults and newborn infants. Isolation by DEAE-cellulose chromatography revealed one main peak (I a) in erythrocytes from adults similar to hexokinase isoenzyme I from rat liver and a smaller peak (I b) which was eluted at a lower conductivity than rat liver isoenzyme II. In erythrocytes from newborn infants the peak I b dominated, isoenzyme I a was the smaller one. In adult erythrocytes an additional isoenzyme was detected which resembled rat liver isoenzyme III, this isoenzyme was absent in erythrocytes from newborn infants. The erythrocytic isoenzymes I a and I b differed in their kinetic properties. Isoenzyme I a had a slightly higher affinity for glucose (Km 47 microM) than isoenzyme I b (Km 100 microM). In the presence of 5mM Pi isoenzyme I b was more inhibited by glucose 6-phosphate than isoenzyme I a. 2.3-Diphosphoglycerate inhibited isoenzyme I b more than isoenzyme I a.