Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the K(m) and k(cat) values of the Dim-5 enzyme for the methylation of a 20 mer peptide to be 7.4 microM and 2.3 min(-1), respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.