Amoebae were isolated from contact lenses of a symptomatic lens wearer in Spain. Protozoa were characterized by studying their morphology, biology, protease activity and the 18S rRNA gene sequence. Morphology of the organism was observed by light microscopy, scanning electron microscopy and transmission electron microscopy. Its structure corresponded to an amphizoic amoeba. The protozoa grew well at 37 °C and poorly at lower temperatures. In addition, it was capable of lysing mammalian cells in vitro. A major 56 kDa proteolytic enzyme was observed in amoeba crude extracts by gelatin–sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Most proteolytic enzymes in protozoa extracts showed significant activity over a wide range of pH (3–9) and temperature (8–45 °C) values. The assays on inhibition of protease activity indicated strongly that enzymes detected in amoeba extracts corresponded to serine proteases and, to a lesser extent, cysteine proteases. The use of proteinase inhibitors on a tissue culture model proved that the proteinase activity is critical for developing focal lesions in HeLa cell monolayers. Finally, partial sequencing of the 18S ribosomal RNA gene and phylogenetic analyses indicated that the isolate is closely related to Acanthamoeba griffini H37 from the UK (T3 genotype).