The oncogenic properties and RNA of the Fujinami avian sarcoma virus (FSV) and the protein it encodes were investigated and compared to those of other avian tumor viruses with sarcomagenic properties such as Rous sarcoma virus and the acute leukemia viruses MC29 and erythroblastosis virus. Cloned stocks of FSV caused sarcomas in all chickens inoculated and were found to contain a 4.5-kilobase (kb) and an 8.5-kb RNA species. The 4.5-kb RNA was identified as the genome of defective FSV because it was absent from nondefective FSV-associated helper virus and because the titer of focus-forming units increased with the ratio of 4.5-kb to 8.5-kb RNA in virus preparations. This is, then, the smallest known tumor virus RNA with a transforming function. Comparisons with other viral RNAs, based on oligonucleotide mapping and molecular hybridization, indicated that 4.5-kb FSV RNA contains a 5' gag gene-related sequence of 1 kb, an internal specific sequence of about 3 kb that is unrelated to Rous sarcoma virus, MC29, and erythroblastosis virus, and a 3'-terminal sequence of about 0.5 kb related to the conserved C region of avian tumor viruses. The lack of some or all nucleotide sequences of the essential virion genes, gag, pol, and env, and the isolation of FSV-transformed nonproducer cell clones indicated that FSV is replication defective. A 140,000-dalton, gag-related non-structural protein was found in FSV-transformed producer and nonproducer cells and was translated in vitro from full-length FSV RNA. This protein is expected to have a transforming function both because its intracellular concentration showed a positive correlation with the percentage of transformed cells in a culture and because FSV is unlikely to code for major additional proteins since the genetic complexities of FSV RNA and the FSV protein are almost the same. It is concluded that the transforming onc gene of FSV is distinct from that of Rous sarcoma virus and other avian tumor viruses with sarcomagenic properties. Hence, multiple mechanisms exist for sarcomagenic transformation of avian cells.