Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400-fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at -80 degrees C. It had an apparent molecular weight of 29,000 +/- 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the KmS for histamine and S-adenosyl-L-methionine were 13.57 +/- 0.74 microM and 6.1 +/- 0.12 microM, respectively; the Ki for S-adenosyl-L-homocysteine was 24.5 +/- 1.45 microM.