Human and rat erythrocytes were fractionated according to change by density-gradient electrophoresis. Some 1-2 ml, or about 1010 of packed cells, can be electrophoresed up a stabilizing sucrose gradient, and require 3-4 h for effective fractionation. The fastest migrating erythrocytes were the least fragile, were larger, incorporated the most 59Fe, and contained more glucose 6-phosphate dehydrogenase activity. These findings are consistent with the most negatively charged erythrocytes being the youngest cells, agreeing with other separative procedures based on density, phase partitioning or serial osmotic hemolysis.