Optimal conditions for mitogen-induced transformation of cynomolgus monkey peripheral blood lymphocytes were determined. Maximal stimulation estimated by the incorporation of tritiated thymidine (3H-TdR) occurred at the concentrations between 25 and 50 μg/ml with phytohemagglutinin (PHA) and concanavalin A (Con A), and between 50 and 100 μg/ml with poke weed mitogen (PWM) in a culture volume of 120 μl containing 2 × 105 mononuclear cells supplemented with 10% fresh autologous serum. Lipopolysaccharide (LPS), however, could induce no significant response. In most animals examined, a high response of blastogenesis was induced by Con A, moderate by PHA, and low by PWM. Optimal stimulation occurred after incubation for 78 hours followed by labeling with 1 μCi of 3H-TdR per culture for 18 hours. Although the extent of blastogenic response was variable in individual animals, the general pattern of time-course and dose-response to mitogens was similar in all monkeys. With 40 monkeys tested, age nor sex did not markedly influence the extent of blastogenic response.