Pili were isolated and purified from Bordetella bronchiseptica. Electron microscopic observations revealed that pili are ubiquitous in this species. The occurrence of pili and flagella appeared to correlate with growth phase and colonial morphology. Pili were about 3 to 4 nm in diameter and morphologically similar to pili isolated from other gram-negative bacteria. Internal core structure was not evident. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified pili showed that up to three different pilus subunit variants could be observed on a single strain, depending on the colonial phase and culture condition. Enzyme immunoassay and immunoblot, however, showed that these subunit variants are serologically related. Mice vaccinated with purified pili were protected against a virulent intraperitoneal challenge of B. bronchiseptica. B. bronchiseptica pili were also found to be similar to Bordetella pertussis pili in morphology and in the molecular size and antigenic structure of pilus subunits. The intact pili of B. bronchiseptica and B. pertussis, however, appeared to have weak serological cross-reactivity.