Cultured endothelial cells from bovine thoracic aortas conditioned with serum-free culture media released an endothelin-1 (ET)-1-like substance. Concentrations of ET-1-like material were determined by bioassay as contractions of isolated ring segments of dog internal mammary vein and by radioimmunoassay. ET-1-like immunoreactivity (ET-1-IR) increased progressively over a 24 h conditioning period and correlated with the bioassay for the first 12 h. Oxyhaemoglobin (1-3 microM) caused a significant two-fold increase in the concentration of ET-1-IR in the medium at 6, 8 and 12 h incubation. Methaemoglobin also caused an approximate doubling of the amount of ET-1-IR at eight h of incubation. NG-Monomethyl-L-arginine (L-NMMA), a blocker of the production of endothelium-derived relaxing factor (EDRF), had no effect on the time-dependent increase in ET-1-IR in the conditioned medium. These results may have important implications for the mechanisms underlying vascular smooth muscle hyperreactivity such as cerebral spasm following subarachnoid haemorrhage.