Rat thymocytes can be separated into two subpopulations by centrifugation for 20 minutes at 1,600 g in an 18/26/36% (w/v) discontinuous gradient of bovine serum albumin. Approximately 13% of the cells band at the 18/26% interface (light cells) while the remaining cells band at the 26/36% interface (heavy cells). In vitro and in vivo studies of 3H-thymidine incorporation indicate that the light cells are 2- to 3-fold enriched in the rapidly dividing lymphoblast subpopulation of thymocytes as compared to heavy cells. Light cells transport the non-metabolizable glucose analogue 3-O-methylglucose (3-MeGlc) approximately four times faster than heavy cells. The time course of 3-MeGlc uptake is biphasic for light, heavy and unfractionated thymocytes. While the half-times of the rapid (1 minute) and slow (20-45 minute) phases of uptake are similar for all three types of cells, the contributions of the rapid phase to total uptake are 50% for light cells, 20% for unfractionated thymocytes and 10% for heavy cells. The results show that 3-MeGlc transport activity differs markedly within certain thymocyte subpopulations. The correlation between the contributions of the rapid phase of uptake and the proportion of lymphoblasts in the thymocyte fractions suggests that the lymphoblast and small lymphocyte subpopulations might be responsible for the rapid and slow phase of 3-MeGlc uptake, respectively.