The N-methylation of pyridine in vitro, using dialysed and undialysed hepatic, pulmonary, renal and brain preparations from rabbits, is described. Analysis of the quaternary metabolite, N-methylpyridinium ion, was carried out by selective ion-pair extraction and cation-exchange high-performance liquid chromatography (h.p.l.c.) using a u.v. detector, and also by direct cation-exchange h.p.l.c. of incubates containing S-adenosyl-L-[methyl-3H]methionine using a flow-through radioactivity detector. N-Methylation of pyridine could be readily demonstrated with dialysed homogenates, 9000 g and 100 000 g supernatant fractions from lung, kidney and liver, but not with any of the brain preparations. 'Pyridine N-methyltransferase' activity was confined to the tissue cytosol, and this enzyme utilized S-adenosyl-L-methionine as the methyl donor. Since the activity of the 'pyridine N-methyltransferase' in rabbit tissues is increased many fold by dialysis, this enzyme, in common with most other N-methylating enzymes, is subject to inhibition by a low-molecular-weight endogenous substance.