A total of 77 field isolates and 15 reference strains of the porcine respiratory pathogen Actinobacillus pleuropneumoniae were tested for their ability to form biofilms in a polystyrene microtiter plate assay. More than half of all field isolates, which included strains representing serotypes 1, 5 and 7, but only two reference strains (serotypes 5B and 11) exhibited biofilm formation. Strains that formed biofilms in microtiter plates also formed thick biofilms at the air-liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth. Our findings indicate that biofilm formation is a prevalent phenotype among A. pleuropneumoniae field isolates, and that this phenotype may have been previously overlooked because of its tendency to be lost upon subculturing in broth. Biofilm formation may have relevance to the colonization, pathogenesis and transmission of this bacterium.