An enzyme-linked immunosorbent assay for the detection of Haemophilus influenzae type b antigen was developed. It was able to detect purified polyribose phosphate at concentrations of ≥1 ng/ml in cerebrospinal fluid. This was 50 times more sensitive than counterimmunoelectrophoresis with the same antiserum. The sensitivity for polyribose phosphate in urine was similar, but that in serum was about 10 times less. Nonspecific reactions were observed with blood-stained cerebrospinal fluid and some sera. These were differentiated from true positive reactions by a blocking test with unconjugated immune serum. A wide range of organisms was tested for cross-reactivity in the assay. With the exception of a protein A-rich strain of Staphylococcus aureus, they gave absorbances of <8% of that of the homologous system. In a series of five cases of proven H. influenzae type b meningitis, the sensitivity of the assay with cerebrospinal fluid was confirmed to be at least 25 times greater than that of counterimmunoelectrophoresis. The results indicate that the enzyme-linked immunosorbent assay is highly sensitive and specific in detecting H. influenzae type b antigen. The necessity to perform the blocking assay on all sera limits its usefulness for the examination of these specimens. However, it should prove valuable for the detection of the antigen in cerebrospinal fluid and urine.