SV40 late replacement vectors containing the polyoma middle-T coding sequences have been constructed. Mixed hybrid virus stocks have been obtained through complementation with a defective SV40 helper genome (dl 1055) following DNA transfection into CV-1 cells. Middle-T antigen is expressed in the infected simian cells at about 5–10 fold higher levels than in polyoma virus-infected mouse cells and has the pp60c-sec-associated tyrosine-specific protein kinase activity in vitro. However, the ‘specific activity’ of the kinase in extracts of the infected CV-1 cells is lower than that observed in polyoma infected 3T6 cell extracts. The half-life of middle-T antigen in the CV-1 cells is about 4 h but the in vitro kinase activity associated with middle-T has a half-life of at least 8 h and hence appears to be stabilized. The in vivo phosphorylated species of middle-T has been shown by sucrose gradient analyses to be largely distinct from the middle-T with associated protein kinase activity in vitro.