CheR methyltransferase from Salmonella typhimurium was directly photolabeled with S-adenosyl-L-[methyl-3H]methionine. The labeled protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then was detected by fluorography. The methylase-S-adenosyl-L-methionine adduct was found to be stable under the experimental conditions employed. Labeling was found to be a function of the concentration of enzyme, S-adenosyl-L-methionine (AdoMet), and the intensity and time of UV irradiation. The extent of labeling and protein methylation was found to be inhibited by S-adenosyl-L-homocysteine, S-adenosyl-L-ethionine, and sinefungin, which are known to compete with AdoMet for the same binding site on the enzyme. Our earlier data showed that the enzyme has 2 cysteine residues and that these are important for enzyme activity. Here, we show that sulfhydryl reagents inhibit the photolabeling of the substrate to the enzyme, indicating the presence of cysteine in the vicinity of the substrate-binding site. We also found that when Cys31 was modified to Ser, no photolabeling of CheR was observed, whereas a modification of Cys229 to Ser had little effect on the ability of AdoMet to label the enzyme. This suggests that Cys31 is located at or near AdoMet-binding site. The labeled protein was cleaved at tryptophan residues, generating two major fragments, each containing 1 cysteine residue. SDS-PAGE and fluorography of the cleaved products indicated the presence of the label being associated with the Cys31 fragment. Similar results were obtained when the labeled protein was cleaved at glutamic acid residues using V8 protease. A tryptic digest of the labeled protein showed two radioactive peptide peaks when subjected to separation on reverse phase high pressure liquid chromatography. The labeled peptides were further digested to free amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that Cys31 may be involved with substrate binding, as well as with catalysis.