Fourteen temperature-sensitive mutants of the Prague strain of Rous Sarcoma virus were isolated. All of the mutants were temperature sensitive for replication, producing infectious progeny 6- to 1000-fold less efficiently than the wild-type virus at the nonpermissive temperature (41.5°) as compared to the permissive temperature (35°). Four mutants were also unable to initiate infection with wild-type efficiency at 41.5°, and all four of these were shown to possess a reverse transcriptase which was more labile than that of the wild-type virus. Two of the other mutants also appeared to map in pol, but expression of the defects in these mutants was most apparent late in infection. Three mutants appear to have mutations mapping in the gag gene, as these mutants produce reduced quantities of [ 35S]methionine labeled virions at the nonpermissive temperature. One mutant was similar to PH734 ( W. S. Mason and C. Yeater, 1977, Virology 77, 443–456), producing virions at the nonpermissive temperature with reduced amounts of the envelope glycoproteins, gp85 and gp37. Four other mutants produced normal yields of [ 35S]methionine-labeled virions at 41.5° containing the viral gag proteins and the α- and β-subunits of reverse transcriptase. The defect in these four isolates has not yet been identified.