To better understand the development of cholinergic-like neurons within the superior olivary complex, we investigated the onset and distribution of two well-known markers of cholinergic-like neurons in hamsters: choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). From embryonic day (E) 14 through postnatal day (P) 0, olivary cells immunopositive for ChAT were restricted to the rostral periolivary (RPO) area. Between P0 and P3, ChAT-positive cells are found in progressively more caudal and ventral periolivary locations. Although rostral and ventral periolivary cells exhibited an early onset of ChAT expression, stable numbers were not reached until P4. In contrast, ChAT expression within the lateral superior olive (LSO) is not visible until after P0 and higher numbers of ChAT-positive cells are obtained by P5. The AChE expression lags several days but follows roughly the same pattern of onset as for ChAT. Additionally in rostral and ventral periolivary regions as well as in the LSO, there were fewer AChE-labeled cells than ChAT-labeled cells. The observed temporal relationships in cholinergic-like expression within olivary cells suggest that different cholinergic-like populations may be defined on the basis of the onset of neurotransmitter-related enzymes: RPO cells are first, cells in ventral periolivary regions are second, and cells associated with the LSO are last. The differences observed in the onset of ChAT and AChE expression may reflect differences in the timing of target innervation as well as differences in synaptogenesis.