Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti-Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 mug. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti-G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge.