Since the localization of nitric oxide synthase (NOS) can be identified by enzyme histochemistry for NADPH-diaphorse (NADPH-d), this method has been used widely for mapping NOS-containing (presumably NOergic) neurons in the central nervous system. So far several studies suggest that NADPH-d is present in distinct neuronal populations in the inferior colliculus (IC), a major processing center for both the ascending and descending auditory pathway, and NO may play an important role in audition. On one hand, there is evidence from several lines of research that the IC makes extensive use of the neuroactive amino acids, in particular the inhibitory transmitter g-aminobutyric acid (GABA) and the excitatory amino acid glutamate (GLU). However, lacking is a description of the distribution of NOergic neurons to which traditional neurotransmitters may be linked. The present research utilized NADPH-d enzyme histochemistry in combination with immunocytochemistry to determine if NO may colocalize with either or both GABA and glutamate in distinct subpopulations of IC neurons. The NADPH-d positive neurons were predominantly found in two main subdivisions of the IC: the external cortex (ECIC) and the dorsal cortex (DCIC). The large numbers of these NADPH-d positive neurons appeared immunostained for GLU while only a small number, seemed to belong to the small cells (somatic area < 100 microm2) similarity to stellate cells group was positive for GABA throughout the cortex of the IC. Owing to no coexistence between GABA and GLU in the same NADPH-d positive neuron in the pairs of adjacent sections of the IC by the mirror-image technique, the present results consequently support that NOergic neurons could be subdivided into at least three distinct populations with a large proportion of about 77% being GLUergic, much lower frequency of about 11% being GABAergic and the remaining 12% expressing non-GABA and non-GLU. In summary, the existence of two functionally distinct populations of NO/GABAergic and NO/GLUergic neurons in the NOergic neurons of IC suggest that at least two differential pattern of GLU-mediated excitatory NO transmission and GABA-mediated inhibitory NO transmission are involved in the networking of auditory communication in the cortex of IC.