A number of viruses which form plaques on the unicellular, eukaryotic, Chlorella-like green alga, strain NC64A, were isolated from fresh water ponds and rivers in Illinois, North Carolina, and South Carolina. The viruses were large polyhedrons (160 to 190 nm in diameter) and contained dsDNA genomes of ca. 300 kbp. All of the viral DNAs hybridized with DNA from the previously described PBCV-1 virus. However, the viruses, even many of those isolated from the same water sample, could be distinguished from one another by DNA restriction endonuclease digestion. The viruses, including PBCV-1, were grouped into five classes by their resistance to certain DNA restriction endonucleases. Presumably the DNAs in the five classes contain different types or amounts of modified bases.