Haemophilus pleuropneumoniae isolates were characterized biochemically, by serotyping, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of enriched outer membrane protein (OMP) preparations. Serotyping of 141 isolates from Iowa and Illinois swine by rapid slide agglutination (RSA) and indirect fluorescent antibody (IFA) tests indicated an overall incidence of serotype 5, 55.3%; serotype 1, 34.0%; serotype 7, 7.8%; and nontypable, 2.8%. There was good correlation between the two test procedures; however, 87.2% of the isolates could be typed by RSA, and only 66.0% by IFA. The profiles of Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae were not affected by colony type, growth medium, time of harvest, or by in vitro or in vivo passage. Seven patterns, based on the apparent mobility of the major OMPs migrating in the 39 to 44 kilodalton (K) region of the gel, a 16 to 16.5K protein, and a heat-modifiable 29K protein, were distinguished among the reference strains of the nine capsular serotypes. Determination of the OMP profiles of 95 field isolates indicated that the H. pleuropneumoniae population is clonal, with three predominant clones distinguished by both serotype and OMP profile responsible for the majority of pleuropneumonia occurring in swine in the United States. Sera from pigs infected with H. pleuropneumoniae serotype 5 were tested for antibodies by immunoblotting with OMP-enriched preparations. Antibodies were detected to all major OMPs of the homologous strain, with the exception of the 42K OMP, and to two broad bands at approximately 54K and 95K. These broad bands did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, were eliminated by periodate oxidation and, thus, were judged to be polysaccharide. Antibodies to the 45K, 49.5K, 66.5K, and high molecular weight (>94K) OMPs, and to the two polysaccharide bands were eliminated by adsorption of sera with whole cells. Antibodies to the heat-modifiable 29/43.5K and the 38.5K OMPs were detected in sera from noninfected pigs. Heterogeneity among serotype 5 strains was apparent in SDS-PAGE profiles of silver-stained, whole-cell, proteinase K-treated lysates and in reactions of sera from infected pigs with heterologous serotype 5 strains. Sera also reacted with OMPs from serotypes 1 and 7; however, several OMPs and polysaccharide determinants of these serotypes appeared to be type specific.