From indirect evidence we have proposed that cholinergic versus non-cholinergic neurons in the laterodorsal tegmental nucleus can be distinguished with the duration of their extracellularly recorded action potentials, "broad" spikes for the former, "brief" for the latter. To test this assumption more directly, we labelled single neurons recorded extracellularly in and around the laterodorsal tegmental nucleus with biocytin or neurobiotin, and processed the sections with reduced nicotinamide adenine dinucleotide phosphate-diaphorase, a proven marker for cholinergic neurons in the laterodorsal tegmental nucleus. Biocytin or neurobiotin which was deposited at the site of recording was incorporated into single neurons. Among 171 trials (91 for broad-spike and 80 for brief-spike neurons), marking was successful in 68 cases (29 for broad-spike and 39 for brief-spike neurons). Almost all (21/22) of the broad-spike neurons located within the laterodorsal tegmental nucleus were positive for reduced nicotinamide adenine dinucleotide phosphate-diaphorase staining, i.e. they were cholinergic, while all of the brief-spike neurons in and outside of the laterodorsal tegmental nucleus lacked the diaphorase activity, and were thus non-cholinergic. The present study shows that, after extracellular labelling of single neurons by biocytin or neurobiotin, cholinergic neurons in the laterodorsal tegmental nucleus are confidently distinguished from non-cholinergic ones in the corresponding area with their spike shapes. It is also shown that the cholinergic neurons distinguished by this criterion are characterized by their tonic firing at slightly lower rate and larger cell size than the brief-spike non-cholinergic ones.