Outer-membrane proteins from isolates of Haemophilus influenzae type b were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarcosinate-insoluble membrane preparations contained one peptide with a molecular weight of 16,000 and four major peptides with molecular weights of 25,000-40,000. A peptide with a molecular weight of 49,000 (50,000 in some strains) was observed after the samples were heated at 100 C. Fifty-one isolates obtained from patients hospitalized with invasive diseases, primarily meningitis, could be subclassified into nine categories based on reproducible and clearly resolvable differences in the outer-membrane protein profiles. Five categories accounted for 92% of the isolates. Complete concordance was observed in subtypes of strains obtained from epidemiologically related cases and contacts. Thus, comparison of the major outer-membrane proteins of H. influenzae type b is a useful technique for investigating the transmission of the organism and may provide a basis for further immunologic characterization of the outer-membrane proteins.