We have developed a radioimmunoassay for big-endothelin-1, based on a new antiserum recognizing human big-endothelin(1-38). On a molar base the antibody showed virtually no cross-reactivity with endothelin-1, -2, -3 (less than 0.001%), 14% cross-reactivity with the C-terminal big-endothelin(22-38) and only 0.85% cross-reactivity with porcine big-endothelin(1-39). However, immunoreactive big-endothelin-1 could be detected in human, rat and rabbit plasma. Thus, big-endothelin-1 in rats and rabbits seem to be structurally similar to human big-endothelin-1. Immunoreactive endothelin-1 was measured using a commercially available anti-endothelin-1 antibody. The measured concentrations of immunoreactive endothelin-1 and big-endothelin-1 and their molar ratios differed significantly in the three species.