The molecular karyotype of 3 clones derived from strain HM1:IMSS of Entamoeba histolytica was studied by transverse alternating field electrophoresis. 11-20 bands ranging between 0.3 and over 3 Mb were resolved. Hybridization with total DNA detected highly repetitive sequences in the slow-migrating molecules, while non-repetitive sequences were located in the intermediate and fast-migrating molecules. rDNA, tubulin, actin, cysteine proteases DNA fragments, and a variable DNA sequence (EhVR1) located the respective genes mainly in the 1.3-1.5-Mb region, although they differed in the three clones. Two-dimensional transverse alternating field electrophoresis showed that more than one high-molecular weight molecule may comigrate in a single DNA band. rDNA, and EhVR1 hybridized with slow-migrating bands in a characteristic ladder pattern. Most of the bands recognized by EhVR1 seems to be linear molecules, although exonuclease III-resistant bands also hybridized with EhVR1, suggesting the presence of circles.