Prolactin (PRL) was extracted with acid-acetone from common carp (Cyprinus carpio) pituitary glands and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography (HPLC) on TSK-gel TMS 250 with a yield of 0.7 mg/g wet tissue. At each stage of purification, fractions were monitored by HPLC on TSK-gel ODS 120T and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Carp PRL was almost equipotent with ovine PRL in retaining plasma Na concentrations in the hypophysectomized killifish, Fundulus heteroclitus. Immunocytochemistry at both the light and electron microscopic levels revealed that carp PRL antiserum specifically stained cells in the goldfish rostral pars distalis. No cross reaction with putative growth hormone (GH) cells in the proximal pars distalis was observed. The specificity of the carp PRL antiserum was confirmed by immunoblot studies. Although immunostaining of both carp and salmon PRL was observed, there was no cross reaction to GHs from these species. Carp PRL had a sole N-terminal residue of valine, a molecular weight of 23 kDa in SDS-PAGE, and an isoelectric point of 7.3 by gel electrofocusing. Based on these results, together with the knowledge of physicochemical properties of salmon and tilapia PRLs, we propose a standard procedure for isolation of fish PRLs.