The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. In the present work, the uptake of rhodamine 123 by individual lymphocytes undergoing blastogenic transformation in cultures stimulated by phytohemagglutinin was measured by flow cytometry. A severalfold increase in cell ability to accumulate rhodamine 123 was observed during lymphocyte stimulation. Maximal dye uptake, seen on the third day of cell stimulation, coincided in time with the peak of DNA synthesis (maximal number of cells in the S phase) and mitotic activity. A large intercellular variation among stimulated lymphocytes, with some cells having fluorescence increased as much as 15 times in comparison with nonstimulated lymphocytes, was observed. Whereas the increased uptake of rhodamine 123 also correlated with the increase in cellular RNA content, the correlation between the dye uptake and cell size (measured by light scatter) was less apparent. As observed by UV microscopy, the increased dye uptake during the blastogenesis was due, to a large extent, to an increase in number of mitochondria per cell. However, an additional increase in rhodamine 123 binding per mitochondrion or per unit of mitochondrial membrane in stimulated cells could not be excluded. The present data indicate that rhodamine 123 may be used as a supravital mitochondrial probe, discriminating between cycling and quiescent cells and having application in sorting functionally distinct cell subpopulations.