Disseminated infections due to microsporidia of the genus Encephalitozoon are detected increasingly, especially in patients with AIDS. Identification of microsporidia can be achieved by a variety of immunological and molecular methods. This study evaluates the feasibility of pulsed-field gel electrophoresis (PFGE) for the analysis of karyotypes of the 3 known species of this genus (Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis) and of 2 of the 3 known E. cuniculi strains (strains I and III). Eleven chromosomal DNA bands were resolved for E. cuniculi and 10 chromosomal DNA bands for E. hellem and E. intestinalis, with molecular sizes ranging from 231 to 320 kb, from 197 to 288 kb and from 195 to 285 kb, respectively, resulting in estimated genome sizes of about 3.0 Mb, 2.5 Mb and 2.4 Mb. Different PFGE chromosomal banding patterns indicate that not only E. cuniculi, as previously described, but also E. hellem, represent a heterogeneous entity. PFGE is a valuable method of evaluating inter- and intra-species variations among Encephalitozoon species that may enable the identification of environmental sources of infection and modes of transmission.