Inoculation of the Soehner-Dmochowski isolate of the Moloney strain of murine sarcoma virus (MSV), designated MSV-SD, consistently leads to the development of bone tumors in the susceptible New Zealand black (NB) rats. Two separate cell cultures have been established from 2 individual MSV-SD-induced NB rat bone tumors. Cells of 1 bone tumor culture, designated RBT-E, are in early in vitro passages. These cells form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at a greatly enhanced rate, 5 times that of normal nontransformed rat embryo cells. Cells of the RBT-E culture release both MSV and murine leukemia virus (MuLV) and therefore contain sarcoma-positive leukemia-positive transformed cells. The other rat bone tumor culture, designated RBT-L, produced MSV at early passages. RBT-L culture has been passaged over 130 times in vitro. Cells of the RBT-L culture form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at an enhanced rate (3 times that of rat embryo cells), indicating the presence of transformed cells within the RBT-L culture. However, cells of the RBT-L culture at late passages (Passage 130 or more) produce only MuLV and no detectable MSV activity (as shown by the lack of tumor-inducing activity and the lack of focus-forming activities by direct assay or by infectious center assay). Attempts to rescue MSV activity from RBT-L cells by cocultivation with MuLV-producing mouse cells were not successful. The MuLV found in the RBT-L cells, however, is a competent helper virus capable of rescuing the MSV genome from MSV-SD-induced hamster bone tumor cells. All the available evidence supports the notion that late passages of the RBT-L culture contain transformed cells that do not produce conventionally detectable MSV. These cells are referred to as sarcoma-negative leukemia-positive cells. The sarcoma-negative leukemia-positive cells represent a different kind of MSV-induced transformed cells and provide a unique system for studies in search of MSV markers such as MSV-specific antigens and MSV-specific nucleotide sequences.