The ocular pathology associated with acanthamoebiasis may result, at least in part, from the excretory and secretory (E&S) products of the active Acanthamoeba trophozoites. To test this hypothesis, the ability of A. polyphaga (ATCC Strain 30461) trophozoite E&S products to digest collagen, the major constituent of the corneal stroma, was evaluated. The secreted proteinases of A. polyphaga were identified using in vitro azocoll degradation, activity-PAGE, radiolabeled extracellular matrix (ECM) degradation, and collagen degradation assays. Inhibitors of serine (phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate), cysteine [benzyloxyphenylalanyl-analyl fluoromethyl ketone, N-ethylmaleamide, ethylenediamine tetraacetic acid (EDTA), L-trans-3-carboxyiran-2-carbonyl-L-leucylagmatine], metallo- (1,10-phenanthroline, EDTA, phosphoramidon), and aspartyl (pepstatin A) proteinases were incorporated into the assays. Proteinase activity was detected in trophozoites and the E&S products of trophozoites but not in cysts. The azocoll and activity-PAGE assays indicated the presence of serine and cysteine proteinases, while degradation of the radiolabeled ECM by live trophozoites confirmed not only the presence of serine and cysteine proteinases but also metalloproteinase(s). Most proteinase activity occurred at neutral pH. Incubation of E&S with type I collagen did not yield the typical 3/4:1/4 products characteristic of vertebrate collagenases. These data suggest that E&S products of A. polyphaga contain multiple serine and cysteine proteinases with nonspecific collagenolytic activity and that metallproteinases form an additional minor constituent.