The presence of catechol-O-methyltransferase (COMT) in the rat brain was studied using a combination of two-dimensional gel electrophoresis (2-DE), protein blotting and a specific antiserum. Two major immunoreactive proteins were identified-one with mol. wt 23 kdalton and an isoelectric point of 5.2, the other of mol. wt 25 kdalton and an isoelectric point of 5.1. In addition, multiple lower molecular weight immunoreactive proteins, possibly corresponding to breakdown products of the enzyme, were also detected. The 23 kdalton form of COMT, which is probably the soluble form of the enzyme, is a major protein visible on silver-stained 2-D gels of rat brain. In contrast, the other proteins recognized by the antiserum were not detected by the silver stain. These results demonstrate, using 2-DE, that at least two distinct forms of catechol-O-methyltransferase are present in rat brain. In addition, since one of these proteins is stained by silver, these results also serve to identify another protein visible on 2-D electrophoretograms of rat brain.