A type II restriction endonuclease, named CviJI, was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA containing virus IL-3A. CviJI is the first restriction endonuclease to recognize the sequence PuGCPy; CviJI cleaves DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. CviJI cleaved DNA into smaller but defined fragments in the presence of ATP. This "star" activity was stimulated by dithiothreitol and/or S-adenosylmethionine but did not occur under conditions which favor "star" activity of other restriction endonucleases.