The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.