Cholinergic transmission in the rat superior olivary complex (SOC) was assessed by densitometric measurements of labeling with several markers: acetylcholinesterase (AChE) histochemistry, choline acetyltransferase (ChAT) immunohistochemistry, and muscarinic acetylcholine receptor (M35) immunohistochemistry and N-methylscopolamine (NMS) binding autoradiography, as well as its subtype 2 (m2) immunohistochemistry. The markers which may occur in cholinergic neurons (ChAT, m2 receptor and AChE) showed dense labeling in the ventral nucleus of the trapezoid body (VNTB), in line with other evidence that it contains cholinergic neuron somata. The markers which are predominantly in cholinoceptive neurons (M35 and NMS) showed less labeling in the SOC, suggesting few muscarinic cholinergic inputs. These results are compared with those for the cochlear nucleus.