From the corneas of nine keratitis patients and from their contact lenses, contact lens boxes and saline solutions, 15 strains of Acanthamoeba have been isolated. An Acanthamoeba strain was isolated from the swimming pool where one of the patients swam, while in the tapwater of the houses of three patients investigated, no Acanthamoeba could be detected. All the Acanthamoeba isolates from the cornea belong to genotype T4, but are different subtypes of T4. The Acanthamoeba detected on the contact lenses (and/or associated paraphernalia) of a patient are of the same subtype as that isolated from the cornea. The only Acanthamoeba strain isolated from a contact lens which was not related to an Acanthamoeba keratitis infection proved to be another genotype. A strain of Hartmannella from a cornea and two vahlkampfiids isolated from contact lenses had no connection with keratitis. This study confirms that, as found elsewhere, only Acanthamoeba genotype T4 of the 12 known Acanthamoeba genotypes is responsible for keratitis in Belgium. Most cases of Acanthamoeba keratitis cases are due to poor hygiene in the treatment (cleaning and storage) of contact lenses.