A highly sensitive and specific enzyme immunoassay (EIA) for the detection of Haemophilus influenzae serotype b antigens in body fluids and broth cultures was developed, with a polyclonal antibody directed against polyribose phosphate as the solid-phase reagent and a biotinylated monoclonal antibody directed against H. influenzae type b outer membrane protein as the liquid-phase reagent. H. influenzae type b antigens could be detected in broth cultures containing as little as 50 organisms per ml. The sensitivity and specificity of this system were compared with those of two commercial kits and counterimmunoelectrophoresis. The overall detection of H. influenzae type b antigens in clinical specimens collected from children infected with H. influenzae type b was as follows: with Phadebact, 86 and 86% in cerebrospinal fluid and urine specimens, respectively; with Bactigen, 86, 80, and 92%, with counterimmunoelectrophoresis, 78, 73, and 75%, and with biotin-avidin EIA, 100, 100, and 100% for cerebrospinal fluid, serum, and urine specimens, respectively. In the biotin-avidin EIA, no positive reactions were noted in specimens collected from patients infected with other bacteria or from patients without evidence of bacterial infection, whereas false-positive reactions were found by counterimmunoelectrophoresis and the commercial kits. These results suggest that this monoclonal antibody reacting with the outer membrane protein is more specific and sensitive than the conventional methods using polyclonal antisera for the detection of H. influenzae type b antigens during severe infections in children.