Seven different forms of the enzyme COMT (catechol O-methyltransferase) were found in isolated rat hepatocytes by two-dimensional gel electrophoresis and immunoblotting: five small variants (S-COMT) and two large variants (L-COMT). The identities of these COMT forms were verified by tryptic fingerprinting using MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, and by amino acid sequencing using ESI-IT-MS/MS (electrospray ionization with ion-trap tandem MS). Analysis of tissue distributions showed that the S-COMT forms were highly expressed in liver and kidney, whereas L-COMT was expressed more strongly in other tissues. Both of the L-COMT forms were found in all of the tissues examined except the heart, which expressed only the most acidic form, and the kidney, which expressed only the most basic form. Subcellular fractionation revealed the presence of both S-COMT and L-COMT in soluble, as well as sedimentable, fractions, suggesting that they should be classified by size rather than (as previously) by localization. Several of the S-COMT forms were N-acetylated, and the two most acidic forms were found to be phosphorylated at Ser(260). One of the latter was unique to liver cells; the other was also found in kidney, brain and thymus. Among the non-phosphorylated S-COMT forms, one was ubiquitous, one was found in testis and liver, and a third was found in liver, kidney and thymus. No other phosphorylated sites were found, suggesting that the pI differences distinguishing between the various COMT forms are due to some as yet unidentified structural modification(s).