An assay for Moloney murine leukemia virus was developed that made use of the production of morphologically altered foci in nonproducer mouse cells (15F) carrying murine sarcoma virus. Wild-type (wt) virus gave a ratio of titers at 39 degrees C/34degrees C = 1.05 +/- 0.45 (standard deviation;n = 20). A spontaneous, thermosensitive (ts) mutant of Moloney murine leukemia virus, ts3, defective in a late viral function, gave 39 degrees C/34degrees C = 0. A murine cell line (TB) was mixedly infected with ts3 and wt (multiplicities of infection, 7.8:4.3), cloned after infection, and shown to be infected by both viruses. At 34 degrees C it produced wt, ts, and particles of mixed parentage. The heterozygotes (hz) had ratios of assays 39 degrees C/34 degrees C = 0.06 to 0.84 (mean, 0.36). To eliminate possible interference by multiploid particles with determination of the proportions of the three types of particles, the virus produced by the mixedly infected, cloned cell line at 34 degrees C was distributed by velocity sedimentation in a sucrose gradient, and virus was picked from the lightest part of the gradient. The proportions of ts, wt, and hz were 0.27, 0.26, and 0.47. Those particles identified as hz segreated ts, wt, and hz in the proportions 0.24, 0.27, and 0.49, respectively. These values were not significantly different from those predicted from a diploid model of the genome.