The lymphocytes of five owl monkeys (Aotus trivirgatus) infected with Herpesvirus saimiri and three control monkeys were studied. Lymphocytes were separated on Ficoll-Hypaque gradients, incubated in suspension cultures, and prepared for electron microscopic and immunofluorescent study at 24, 48, and 72 hours after the beginning of culture. Buffy coats of whole blood obtained immediately after bleeding were also prepared for study. At the time of the study, four of the five infected monkeys had died with malignant lymphoma and lymphocytic leukemia. Herpesvirus saimiri virions were demonstrated by electron microscopy and H saimiri antigens by immunofluorescence in 1-20% of the lymphocytes from infected monkey in two of five cultures at 24 hours after culture, four of five at 48 hours after culture, and all of five at 72 hours after culture. There was good agreement between the electron microscopic and the immunofluorescent data. None of the control monkey cultures and none of the buffy coat preparations contained H saimiri virions or antigens. By electron microscopy, the great majority of the virus particles were nucleocapsids within the nuclei of lymphocytes. Enveloped virions were seldom observed. There was some evidence to suggest that the higher the percentage of lymphocyte containing H saimiri, the poorer the prognosis for the monkey. Herpesvirus saimiri was isolated from all five infected monkeys by co-cultivation of lymphocytes with Vero cells. Control lymphocyte co-cultivations were negative.