The concentrations of vasoactive intestinal polypeptide (VIP) and the peptide with NH2- terminal histidine and COOH-terminal isoleucine (PHI) in various peripheral tissues and some areas in the CNS of the cat were compared with their immunohistochemical localization. The VIP levels in the gastrointestinal tract were 3 to 6 times higher than PHI levels. Much (up to 10-fold) higher VIP than PHI levels were also observed in the genitourinary tract as well as in the lung and heart. In the neurohypophysis, however, the VIP/PHI ratio was close to 1. Gel-permeation chromatography revealed that VIP- and PHI-immunoreactivity (IR) in the intestine, pancreas and brain consisted of three larger molecular forms in addition to the 'standard' peptides. These larger forms which had overlapping elution positions may represent prepro-VIP/PHI forms. The immunohistochemical analysis revealed that VIP- and PHI-IR was present in the same ganglion cells in the intestine, pancreas, uterus and sympathetic ganglia. Furthermore, the terminal networks for these two peptides were very similar in the periphery. In the median eminence of the hypothalamus and in the posterior lobe of the pituitary, considerably more nerves were PHI- than VIP-IR. This observation was in parallel to a low VIP/PHI ratio. In conclusion, VIP and PHI seem to co-exist in most neuronal systems. Although the ratio of VIP and PHI on the precursor gene is 1:1, differences in posttranslational processing may create a considerably higher content of VIP than PHI in most terminal areas.