Sarcoma virus-positive, leukemia virus-negative (S + L −) mouse cells transformed by defective but rescuable murine sarcoma virus (MSV) release noninfectious type C virions with a quantitative deficiency of viral-type RNA-dependent DNA polymerase (RDDP). Human S + L − cells containing the same genome fail to express any detectable viral-type reverse transcriptase. To study this difference in MSV expression, second-generation MSV from the S + L − human cells is recloned in dog, mink, and back into mouse cells. Heterologous host dog and mink S + L − cell clones, like human S + L − cell clones, fail to release viral-type reverse transcriptase into culture supernatant fractions. All second-generation homologous mouse S + L − cell clones again release viral reverse transcriptase in supernatant fractions, indicating that the MSV genome has not been altered in this function. Using (dT) 12–18-cellulose and phosphocellulose chromatography of cellular polymerase preparations, no intracellular buildup of unreleased murine reverse transcriptase is detected in the S + L − clones. The data presented here suggest that the MSV genome is defective in the information for the viral core protein RDDP. The implications of these findings for the genetic study of MSV are discussed.