The age-associated decline in T lymphocyte function can be attributed in part to shifts between subsets (in particular the accumulation of memory T cells at the expense of naive T cells), and in part to alterations of function within the different T lymphocyte subsets. We show that the fluorochrome rhodamine-123 (R123) can distinguish further subdivisions within the naive and memory populations of both CD4 and CD8 T cells in the mouse. Aging leads to a progressive increase in the proportion of T cells in each subset that stain dimly with R123, and a corresponding decrease in the proportion of R123hi T cells, which are typical in young mice. We show that differential R123 staining between T cell subsets is due to variation in the activity of P-glycoprotein (P-Gp), the 170 kDa, ATP-dependent plasma membrane pump that mediates multiple-drug resistance. All T cells initially take up equivalent amounts of R123, but some T cells then can extrude the dye in a temperature-dependent process that is blocked by each of four pharmacologically disparate inhibitors of P-Gp function. Immunofluorescence experiments suggest that T cells from old and young mice have equal amounts of the P-Gp molecule itself, suggesting that differences in R123 extrusion may depend on posttranslational regulation of P-Gp activity.