In SV40-transformed or infected rat cells phosphorylation of the tumor suppressor protein p53 is enhanced due to activation of kinases. At least three different kinases can be co-precipitated with p53-large T (LT) immune complexes, casein kinase II representing the major activity, a cyclin dependent kinase (Cdk), and a kinase which appears to be specifically activated by LT (E Müller, B Boldyreff and KH Scheidtmann, Oncogene 8: 2193-2205, 1993). In this paper we describe the purification and identification of the LT-activated kinase that phosphorylates a site adjacent to the Cdk site in rat p53. To monitor the activity a synthetic peptide was used containing glutamic acid at the position of Ser-313, thus mimicking a phosphorylated Cdk site. With a combination of Mono Q chromatography and subsequent affinity chromatographies with p13suc1 and a p53-fragment as ligands a 42 kDa protein kinase was purified to near homogeneity from SV40-transformed rat cells. This kinase phosphorylated both the peptide substrate and the native rat p53. Interestingly, phosphorylation of the specific site seemed to depend on prior phosphorylation of the Cdk site. On the other hand, the kinase seemed to be activated by LT, as the activity towards the peptide substrate was significantly higher in extracts from wild-type LT-transformed cells than from normal or mutant LT-transformed cells. This activation was not restricted to rat cells but occurred in SV40-transformed mouse and infected monkey cells as well. Phosphorylation of the specific site by LT-activated kinase was not dependent on the presence of LT in vitro suggesting that activation of the LT-activated kinase is probably indirect rather than through direct interaction with LT. Cell cycle studies revealed that the LT-activated kinase is cell cycle regulated, since its activity was not detectable in M phase but increased during G1 phase after which it remained relatively constant.