In a previous paper [Lim, Park, Jee, Lee and Paik (1999) J. Cancer Res. Clin. Oncol. 125 , 493Ő499], we showed two major forms of active DNA-6- O -methylguanine:protein- L -cysteine S -methyltransferase (MGMT; EC 126.96.36.199) in the liver with N -nitrosodiethylamine (DEN)-induced carcinogenesis: these were 26 and 24kDa species. Here we show that a 2kDa C-terminal fragment was cleaved from the 26kDa species in vitro by thrombin or microsomal fractions isolated from DEN-treated rat livers. When Ser 204 of the 26kDa protein was replaced with Ala by site-directed mutagenesis, phosphorylation of the protein was completely abolished, indicating Ser 204 to be the site of phosphorylation. We also show that the phosphorylation was performed by Ca 2+ -independent protein kinase isoenzymes, and that the phosphorylated rat MGMT protein was resistant to digestion by protease(s) whose activity was increased during DEN-induced hepatocarcinogenesis and also by digestion with endopeptidase Glu-C (V8 protease).