Short-term lymphocyte cultures from mouse thymus, spleen, or lymph nodes were studied for the presence of murine leukemia virus group-specific antigens with an immunofluorescence test using rat immune sera against syngeneic cells infected with the radiation leukemia virus or its pseudotype of murine sarcoma virus and goat and rabbit antisera against purified murine leukemia virus group-specific antigen. Antigens reacting with these sera appeared in the cultured lymphocytes within 24 hr, and the proportion of immunofluorescent-positive cells increased to 25-80% by the second or third day of cultivation, concomitantly with a decrease in cell viability. The appearance of these antigens can be suppressed by inhibitors of DNA (mitomycin-C), RNA (actinomycin-D, cordycepin, and polyadenylic acid), and protein (cycloheximide) synthesis. No infectious virus could be detected by the immunofluorescence and XC-cell tests. The observed phenomenon appears to represent the spontaneous partial derepression of endogenous murine leukemia virus replication in lymphocytes during short-term in vitro cultivation.