Canine blood lymphocytes were nonlytically separated on antibody-coated petri dishes into surface immunoglobulin-positive (SIg+) and -negative (SIg-) populations. SIg- cells were further separated into cells reactive or non-reactive with monoclonal antibody DT-2 recognizing canine T lymphocytes. The purity of the three enriched lymphocyte populations exceeded 90% as assessed by immunofluorescence. Mitogen stimulation showed a vigorous response of SIg+ cells to pokeweed mitogen and concanavalin A but only a weak response to phytohemagglutinin. In mixed DT-2- and DT-2+ cells responded to phytohemagglutinin, concanavalin A and pokeweed mitogen, and both populations were good responders in mixed leukocyte culture. Only DT-2- cells were potent stimulators; DT-2+ cells were not. Hence, canine blood T cells can be divided into two subsets, DT-2+ and DT-2-, both of which are responsive to mitogens and alloantigens.