Lymphocytes obtained from cardiac blood and thoracic duct lymph of the opossum (Didelphis virginiana) were incubated in short-term culture with phytohemagglutinin. Relatively pure inocula of lymphocytes were prepared through elimination of erythrocytes and granulocytes. A critical condition for successful culture growth was found to be the use of heat-inactivated rat serum in place of autologous or isologous sera. Because of rapidly occurring bursts of DNA synthesis, cellular transformation and mitotic activity, cultures were harvested at one half the incubation time common to human blood cultures and those of other laboratory animals. Opossum lymphocytes provide excellent material for cytogenetic investigations because of this short incubation requirement and because their chromosomes are large and few in number. Particularly significant is the ease with which the sex chromosomes may be identified.