Murine lymphocytes cultured with frozen and thawed Rauscher murine leukemia virus 100,000 x G supernatants demonstrated increased 3H-thymidine uptake. The stimulatory capacity co-purified with the major envelope glycoprotein, gp70, and can be specifically removed from viral supernatants by absorption with anti-gp70 antibody-linked Sepharose. Cells from all strains tested, including strains prone to autoimmune disease (MRL/1, (NZB X W)F1, NZB, and BXSB), immunologically normal strains (DBA/2, C57B1/6, BALB/c, C3H and 129/J), genetic low responders to LPS (C3H/HeJ), and mice congenitally T cell deficient (BALB/Wehi nu/nu) were equivalently responsive to viral protein stimulation. The response peaked after 3 to 4 days of culture, and cells responded at that time after brief exposure to virus supernatants at the initiation of culture. When gp70 concentrations comparable to serum levels were used, both spleen and mesenteric lymph node cells were capable of responding with stimulation indices of between 5 and 10; whereas bone marrow cells responded poorly, and neither thymocytes nor cortisone-resistant thymocytes responded. Lymphocyte stimulation was not diminished by the depletion of adherent cells, and both T- and B cell-enriched populations responded.