The cholinergic system in the rat superior olivary complex (SOC) was evaluated by immunohistochemistry for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and histochemistry for acetylcholinesterase (AChE). ChAT-positive somata were found mostly in the lateral superior olive (LSO) and ventral nucleus of the trapezoid body (VNTB). In the LSO, there were both rostral-caudal. and medial-lateral gradients in concentration of ChAT-positive somata; the highest concentration was in the middle of the rostral-caudal extent and the most medial part. The estimated total number of ChAT-positive neurons in the LSO was similar to previous estimates of the total number of lateral olivocochlear neurons. Two groups of ChAT-positive somata were found in the VNTB: a dorsolateral group of larger, multipolar, and more darkly labeled neurons and a ventromedial group of smaller, oval, and more lightly labeled neurons, which was about 5 times as numerous. There was a caudal-to-rostral increase in number of neurons in each group. VAChT immunoreactivity, predominantly localized in puncta, was seen in LSO, VNTB, and LNTB, and, to a lesser extent, in other parts of the SOC, VAChT-positive somata were also found in the VNTB and medial LSO, This distribution pattern of VAChT was generally similar to that of ChAT. AChE labeling had a similar appearance to ChAT labeling in the VNTB but differed in the LSO, where AChE labeling was lighter and associated more with neuropil than with somata. (C) 1998 Wiley-Liss, Inc.