A direct and simple plaque assay has been developed for the titration of avian leukosis virus. Under optimal conditions macroscopically visible plaques appeared 7 days after infection. The plaque-forming agent belongs to the group of avian RNA tumor viruses as demonstrated by its density, reverse transcriptase activity, polypeptide composition, and by its serological properties. Screening of a series of different strains, including nontransforming avian sarcoma virus mutants, revealed that the assay is applicable to a variety of leukosis viruses; however, so far it has been limited to members of subgroups B and D. Foci induced by sarcoma viruses were also detected as “plaques” when stained with neutral red late after infection. Again, subgroup specific differences were observed between several strains. The assay described permits the determination on the same dish of the ratio of sarcoma and leukosis viruses in virus stocks. It has been used to confirm that several strains of Rous sarcoma virus (RSV) contain associated viruses in excess. In addition, nontransforming plaque-forming viruses were found in varying proportions in stocks of clone-purified helper-independent strains of avian sarcoma viruses.