Neurokinin A and B (NKA and NKB) and neuropeptide K (NPK) were recently isolated from porcine spinal cord and brain, and together with substance P (SP) considerably extend the list of tachykinin-like peptides present in the mammalian nervous system. In order to investigate the distribution of tachykinins in the central nervous system (CNS) we have recently developed sensitive radioimmunoassays (RIA) for both SP and NKA. As NKB and NPK cross-react in the RIA for NKA we were able to determine the content of NKA, NKB and NPK after separation of rat CNS extracts by reverse-phase high-performance liquid chromatography (HPLC). Comparison of different extraction methods suggested that 0.1 M hydrochloric acid gave the best extraction and recovery of NKA-like immunoreactivity from rat brain. Characterization of these tachykinins using HPLC and gel-filtration columns revealed that C18 columns did not adequately separate NPK from NKB under the conditions used by previous authors. Thus 'NKB' content reported previously on the basis of HPLC separation would correspond to the sum of both NPK and NKB content. In the present study, therefore, we introduced modified elution conditions to resolve NPK from NKB and determined the regional distributions of these tachykinins in the rat CNS. SP was the most abundant tachykinin in every region studied. After SP, the NKA concentration was highest and NKB concentration was lowest in all regions except for the cortex and hippocampus where the NPK concentration was highest. The molar ratio of these peptides seemed to be relatively constant in the 3 regional groups (striatum-substantia nigra, cerebral cortex-hippocampus, dorsal root ganglia-dorsal and ventral horns of spinal cord) and suggests that regional specific translation or processing may exist.