Galanin is a regulatory peptide with wide distribution in the central and peripheral nervous system and with numerous biological effects. Several radioimmunoassays based on antisera raised against porcine galanin have been used to measure immunoreactivity in rat tissues. However, considerable lack of parallelism has been observed between the porcine standard and rat tissue extracts, which may decrease the reliability of the quantitative data. The purpose of the present study was therefore to raise antibodies against rat galanin and establish a competitive radioimmunoassay for rat galanin. Two antisera, RatGal4 and RatGal5, were characterized in detail. The homogeneity of the immunoreactive material from several tissues was also investigated with column chromatography. At reverse-phase high-pressure liquid chromatography more than 95% of the immunoreactive material from rat CNS eluted as a single peak in the position of synthetic rat galanin, whereas almost half of the immunoreactive material from the intestine eluted in positions different from the synthetic peptide. Extracts of rat brains as well as jejunum diluted in parallel with the standard curve for both antisera. We conclude that measurements of rat galanin based on these antisera are therefore more reliable than those based on antisera raised against porcine galanin.