Bordetella bronchiseptica has the ability to invade and survive intracellularly. This potential to survive for extended periods within eukaryotic cells might play an important role in the pathogenesis of the infections caused by this microorganism. The bacterial factors involved in this process, however, have not yet been determined. In this study we have identified an acid phosphatase produced by B. bronchiseptica, but not by other Bordetella spp. The expression of this enzyme was demonstrated to be strictly regulated by temperature (optimal expression at 30 degrees C) and seems to be partially repressed by the product of the bvg locus. The enzyme was localized in the cytoplasmic fraction, the optimal activity was observed at pH 5.5, and the apparent molecular mass obtained by zymogram was 40 kDa. To further investigate the pathogenic role of this enzyme, mutants lacking acid phosphatase activity were obtained from both bvg-positive and bvg-negative parental strains using minitransposons. The growth pattern of these recombinant clones in vitro was similar to the parent strains, however, the tested clones exhibited a significant reduction (P < or = 0.05) in their intracellular survival ability. This newly described acid phosphatase from B. bronchiseptica seems to play a role in intracellular survival, and therefore represents a novel pathogenicity factor.