Using highly specific and sensitive radioimmunoassays (RIAs) for endothelin-1 (ET-1) and endothelin-3 (ET-3), we have determined the concentrations of ET-1- and ET-3-like immunoreactivities (LI) in human cerebrospinal fluid (CSF). The concentration of ET-3-LI (82.5 +/- 4.8 pg/ml) was about 1.5-fold greater than that of ET-1-LI (34.3 +/- 1.3 pg/ml). There was no significant correlation between ET-1-LI and ET-3-LI concentrations in human CSF (r = 0.44). Reverse-phase high-performance liquid chromatography (HPLC) revealed a single major component of ET-1-LI and ET-3-LI coeluting with authentic ET-1 and ET-3, respectively. It is suggested that ET-1 and ET-3 play a neuropeptide-like role in the central nervous system.