A type II restriction enzyme (Tha I) has been isolated from the thermophilic mycoplasma Thermoplasma acidophilum. A new method of general application was used to determine the DNA sequence cleaved by the enzyme. Tha I cuts DNA in the centre of the sequence CGCG. Single-stranded DNA is not a substrate. Tha I does not cut T. acidophilum DNA which is presumably modified. This is the first description of a restriction enzyme from a mycoplasma. Because Tha I is easily prepared in large amounts of approximately 10(5) units per gram of cells, it will be a valuable addition to the battery of restriction enzymes used in studies of DNA sequences. It is active at high temperatures and may therefore be useful for special purposes requiring more extreme conditions.