Binding sites for calcitonin and calcitonin gene-related peptide are widely distributed in the central nervous system. In this study, binding of [(125)I]-alpha-rat calcitonin gene-related peptide and [(125)I]-salmon calcitonin in adjacent sections of rat brain revealed clearly distinct patterns of binding in most regions although in some restricted areas such as parts of the ventral striatum, including the nucleus accumbens, there was some overlap in the patterns of binding. In the primary olfactory cortex, which bound only calcitonin gene-related peptide, salmon calcitonin was very weak in inhibiting the binding of calcitonin gene-related peptide. In the nucleus accumbens, high affinity binding of calcitonin and calcitonin gene-related peptide at their homologous receptors was observed, with affinity constants for calcitonin and calcitonin gene-related peptide of 1.4 x 10(9) M(?1) and 1.2 x 10(9) M(?1) respectively. Cross competition studies in this nucleus demonstrated that salmon calcitonin was able to compete for [(125)I]-rat calcitonin gene-related peptide labelled sites with high affinity, with an affinity constant of 0.8 x 10(9) M(?1). However, rat calcitonin gene-related peptide was less potent in inhibiting the binding of [(125)I]-salmon calcitonin labelled sites with only 28% inhibition at 10(?6)M. Further characterization of the calcitonin sensitive calcitonin gene-related peptide labelled sites demonstrated that a range of calcitonin analogs inhibited the binding of [(125)I]-rat calcitonin gene-related peptide with the same order of potency as the analogs competed for [(125)I]-salmon calcitonin labelled sites. Digital substraction mapping revealed calcitonin-sensitive calcitonin gene-related peptide binding sites over parts of the ventral striatum, including mid-caudal nucleus accumbens and fundus striati; over the lateral border of the lateral bed nucleus of the stria terminalis; part of the central amygdaloid nucleus; the organum vasculosum of the lamina terminalis and area postrema and over the wings of the dorsal raphe. These results demonstrate the existence of a new subtype of calcitonin/calcitonin gene-related peptide binding site, which has high affinity for the two otherwise biochemically distinct peptides.