Naegleria fowleri is the etiologic agent of primary amebic meningoencephalitis, a rare but rapidly fatal disease of humans. It invades the central nervous system via nasal mucosa and cribriform plate. Once in brain tissue, the organism induces an acute hemorrhagic, necrotizing meningoencephalitis. We hypothesize that a protease released by the parasite contributes to tissue destruction and facilitates host invasion. Analysis of conditioned media of N. fowleri cultures revealed a major 30-kDa protease with substrate and inhibitor specificity consistent with cysteine proteases. Amino-terminal amino acid sequence of the purified enzyme showed it to be a thiol protease with homology to cathepsin L. It catalyzed the in vitro degradation of extracellular matrix and had a cytopathic effect on mammalian cells. Both ameba-induced matrix degradation and the cytopathic effect are inhibited by Z-Phe-Ala-fluoromethyl ketone, an irreversible cysteine protease inhibitor. Our results indicate that N. fowleri secretes a cysteine protease with the capacity to destroy host tissue. Naegleria gruberi, a nonpathogenic species, expresses a similar protease but, unlike its pathogenic relative, is not thermotolerant to temperatures above 30 degrees C.