Canine lymphocytes from peripheral blood, lymph nodes, thymus and bone marrow were stimulated with phytohemagglutinin-P (PHA) or concanavalin-A (CON-A) to form colonies in methylcellulose. Lymphocytes exposed to mitogens in liquid phase formed clumps the size of colonies. Lymphocyte clumping was eliminated by plating cells directly into methylcellulose, but high concentrations of mitogens (CON-A or PHA is greater than 10 mg/10(6) lymphocytes) were required in order to get subsequent colony formation. Thus, in contrast to published reports, exposure of lymphocytes to mitogen prior to plating was not required for cloning of canine peripheral blood lymphocytes. Colonies from thymus, lymph node, or peripheral blood consisted predominantly of T lymphocytes, whereas cultures from bone marrow also produced colonies with macrophage morphology and surface-adherent colonies with mesenchymal morphology.