Immunohistochemical methods were used to visualize choline acetyltransferase (ChAT)-, serotonin-, dopamine-beta-hydroxylase (DBH)-, and tyrosine hydroxylase (TH)-containing fibers in the primary auditory cortex of the cynomolgus monkey (Macaca fascicularis). Each antiserum revealed a subpopulation of axons with a distinct density and laminar distribution. ChAT-immunoreactive fibers were very dense in superficial layers, particularly in layers I, deep III, and IV, and very sparse in layers V and VI. No immunoreactive cell bodies were evident. Serotonin-immunoreactive fibers were very dense in all cortical layers but exhibited some subtle laminar differences in fiber size and orientation. The densities of DBH- and TH-immunoreactive fibers were substantially lower than the densities of both ChAT- and serotonin-immunoreactive fibers, particularly in layer IV. However, there were substantial differences between the distribution of TH-immunoreactive fibers and that of DBH-immunoreactive fibers. For example, the density of TH-immunoreactive fibers was substantially greater than that of DBH-immunoreactive fibers in layer I. In addition, TH-immunoreactive fibers differed from the other three systems in that TH-immunoreactive fibers exhibited a rostral to caudal gradient of decreasing density. This is the first characterization of the innervation of a specific cortical region by all four of these systems and the first detailed description of the cholinergic innervation of a primate neocortical region utilizing a specific anti-ChAT antiserum. These striking differences in density and laminar distribution suggest that the subcortical extrathalamic systems furnishing these axons differ significantly in their modulation of cortical auditory processing. These data extend observations of previous studies which revealed that the expansion and specialization of the primate neocortex is accompanied by a pronounced regional and laminar differentiation in the intracortical distribution of these highly divergent, extrathalamic afferents.