We have prepared 16 anti-bilirubin monoclonal antibodies and described their unique reactivity to bilirubin and related compounds. Using the modified mixed anhydride method, bilirubin was covalently coupled to bovine serum albumin. We performed somatic cell fusion between murine spleen cells immunized with this bilirubin-albumin complex and murine myeloma P3-X63-Ag8-U1 cells. After screening assays, 16 clones were identified which were producing antibodies not to albumin but to haptenic bilirubin. In inhibition analysis, the antibodies in the culture supernatants cross-reacted with bilirubin glucuronides to varying degrees, but rarely reacted with structurally related biliverdin, hemin, and azodipyrroles of bilirubin. Albumin, when present in the solution, much reduced the reactivity of several monoclonal antibodies to unconjugated bilirubin, and this effect was partly reversed by addition of salicylate which dissociates the binding between bilirubin and albumin.