Guinea pig T lymphocytes have receptors of different affinity for rabbit red blood cells (RRBC): those binding RRBC immediately are termed "active" T cells; the remainder, which bind RRBC only after longer incubation times, are "non-active" or "late-rosetting" T cells. We have found that these two subpopulations have different functional characteristics. Active T cells could not be stimulated effectively with phytohemagglutinin (PHA), and stimulation with concanavalin A (ConA) increased their DNA synthesis only at high concentrations. The non-active subpopulation responded better to PHA but poorly to ConA. Unseparated (total) T cells, however, responded well to both mitogens, suggesting a helper effect by the active T cells. The presence of monocytes in T-cell cultures further enhanced mitogen-induced DNA synthesis. Active T cells were not present in guinea pig bone marrow, whereas they constituted 10% of all lymphocytes in the thymus, 13% in the spleen, 29% in lymph nodes, and 32% in the peripheral blood. After administration of antigen in vivo, the number of active T cells in the regional lymph node increased, whereas the number of total T cells did not change. The similarity of the active T cell populations in guinea pigs and humans increases the usefulness of these animals for preclinical tests of potential new immunomodulating agents.