Abstract The gag-mos hybrid protein encoded by ts110 MoMuSV was shown to have an associated protein kinase activity which phosphorylated both P85 gag-mos and P58 gag when [ γ- 32P]ATP and a manganese cofactor were added to an immune complex containing P85 gag-mos . Immunoprecipitation and removal of P85 gag-mos from the reaction mixture by either an anti- mos or anti- gag serum resulted in a subsequent elimination of in vitro P85 gag-mos and P58 gag phosphorylation. This kinase activity was shown to be either an intrinsic property of P85 gag-mos or else a tightly bound cellular enzyme activity resistant to elution with 2.0 M NaCl, 0.5% deoxycholate, and 0.1% SDS. A correlation was made between the amount of kinase activity and the concentration of P85 gag-mos . Viral gag antisera were also used to show immune complex phosphorylation of another gag-mos hybrid protein termed P100 gag-mos , derived from a revertant of ts110. In vitro phosphorylation experiments derived from v- mos transformed MuSV 124 cells using viral gag antisera were completely negative which shows that the gag-mos kinase in 6m2 cells is not merely a gag-associated kinase that phosphorylates MuSV coded gag gene products. When shifting 6m2 cells from a permissive temperature to the nonpermissive temperature of 39° for 2–4 hr, a noticeable change toward a more normal morphology occurs. NRK 54-5A4 cells infected with a revertant of ts110 with wild-type phenotype, showed little change in morphology between permissive and nonpermissive temperatures. In addition to the is defect affecting P85 gag-mos production previously reported, a second is defect in ts110 is reported here which is functional in nature; it can be detected within 5 min after shift to 39° by the heat lability of the P85-associated kinase activity. The P100 gag-mos protein kinase from the wild-type revertant cells did not exhibit this heat sensitivity under similar conditions. The thermal inactivation of the P85 kinase was shown to precede events that occur as cells are shifted to the restricted temperature including morphological reversion to the normal phenotype, and the decrease in P85 gag-mos concentration. Based on all of these observations, it is suggested that the P85-associated kinase activity is not merely an adherent cellular kinase, but actually a function of the gag-mos gene product.