In our previous in situ hybridization study in the adult rat spinal cord we demonstrated that only neurons express mRNAs for acetylcholinesterase (AChE; EC 3.1.1.7.) and butyrylcholinesterase (BuChE; EC 3.1.1.8.) in this CNS region; no staining was detected for these two messages in the glia cells. Here we used immunocytochemical staining for choline acetyltransferase (ChAT; EC 2.3.1.6.) to answer the, as yet only briefly approached, question whether the cells expressing the messages for both cholinesterases are cholinergic. All neurons expressing mRNAs for AChE and BuChE were found to be ChAT positive. This finding supports the previous proposal that BuChE modulates cholinergic transmission in AChE knockout mice.