Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases.