Cysteine proteinases have a number of important functions in the life cycle of protozoan parasites. Based on our previous studies demonstrating the role of cysteine proteinases in invasion by Entamoeba histolytica, we evaluated the cysteine proteinases of E. invadens, a related protozoan which causes invasive disease of reptiles. E. invadens readily encysts in axenic culture and provides a model to investigate the role of cysteine proteinases in encystation. Broad bands of approximately 130-230, 55, and 35 kDa were detected on gelatin substrate gels and were inhibited with specific cysteine proteinase inhibitors. Maximal enzymatic activity was detected with peptide substrates containing arginine in the P2 position. A 567-bp fragment containing the active site of an E. invadens cysteine proteinase gene was amplified by PCR and had 37.7, 79.1, and 67.9% identity to the derived amino acid sequences of the acp 1, 2, and 3 genes, respectively, of E. histolytica. The PCR product hybridized with a single band of 1.1 kb on a Southern blot of EcoRI-restricted E. invadens genomic DNA. Long-term inhibition of cysteine proteinase activity during encystation resulted in significantly fewer cysts (P < 0.02); however, this effect appeared to be secondary to decreased trophozoite cell division. No difference in chitin synthase activity was detected between controls and encysting cells with inhibited cysteine proteinases, suggesting that these proteinases are not critical for activation of a zymogen form of chitin synthase. These studies demonstrate that cysteine proteinases may be critical for the survival of E. invadens, and specific inhibition may ultimately interrupt transmission.