The carboxyl-terminal fragment of CENP-B contains a major epitope for anti-centromere antibodies (ACA). We have developed an enzyme-linked immunoassay (ELISA) for measuring antibodies to the 147-carboxyl-terminal amino acids of CENP-B expressed as a beta-galactosidase fusion protein. The ELISA was 98% sensitive and 95% specific for detecting ACA in a population which included 46 patients with ACA detected by other means. Therefore, the CENP-B ELISA should prove a valuable tool in screening for ACA in populations at risk of developing systemic sclerosis, such as those with Raynaud's phenomenon. Levels of anti-CENP-B antibodies were not increased in unaffected relatives of probands with ACA.