The macronuclear DNAs from 20 different species of Tetrahymena were characterized using Alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100-2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species of Tetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species of Tetrahymena based on this hybridization pattern parallels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains of Tetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.