We showed recently that human O(6)-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282-287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg(2+) more efficiently than Mn(2+) for phosphorylating human recombinant AGT (rAGT) protein. Both [gamma-(32)P]ATP and [gamma-(32)P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130 kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130 kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.