Abstract A new, simple, and rapid assay method for O 6-methylguanine–DNA methyltransferase (MGMT) has been developed. When [ methyl- 3H] DNA radiolabeled with N-[ methyl- 3H]- N-nitrosourea was incubated together with tissue homogenate, [ methyl- 3H] group was transferred to the enzyme, forming S-[ methyl- 3H]cysteine. In contrast to the previous methods which determined the amount of [ methyl- 3H] group removed from [ methyl- 3H] DNA, the present method measured the amount of [ methyl- 3H] transferred to the enzyme. This has been done by hydrolyzing the radiolabeled enzyme with pronase which is a proteolytic enzyme with a broad substrate specificity. On pronase digestion, [ methy- 3H]-labeled enzyme becomes soluble in trichloroacetic acid. The method is very simple and rapid, and the only expensive equipment required is a scintillation counter which is a relatively routine piece of equipment at present. More than a dozen samples can be processed within 4–5 h without any difficulty. This new method has been employed in the studies on organ distribution of MGMT of rat and mouse.