Varying the concentration of Triton X-100, a nonionic detergent used to promote the DNA polymerase activity of Rous sarcoma virus in an endogenous reaction, showed a very sharp peak at about 0.02% (vol/vol) for optimal DNA synthesis. The yield of DNA at this concentration of Triton exceeded yields obtained at concentrations above the optimum by a factor of 2-5 for the 90-min reaction. At optimal Triton concentration, about 1-7% of the DNA made in the absence of actinomycin and about 4-10% of the DNA made in the presence of actinomycin was 2.5 X 10(6) daltons or greater, as estimated by formamide polyacrylamide gel electrophoresis and by alkaline sucrose gradient sedimentation. No large DNA was obtained at higher than optimal Triton concentrations. The large DNA molecules were rendered totally resistant to single-strand specific nuclease S1 after hybridization to an excess of viral RNA. It was concluded that at optimal detergent concentration, the viral DNA polymerase can synthesize full-size DNA transcripts of viral RNA.