A new sequence specific endonuclease, Mra I has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage lambda DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on phi X174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage lambda DNA are different from those cleaved by Sma I, Xma I and Xor II. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of lambda DNA. Mra I is shown to be an isoschizomer of Sac II and Sst II recognizing the palindromic nucleotide sequence '5-CCGC reduced GG-3'. The enzyme shows an absolute requirement of Mg2+, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45 degrees C and pH 7.0. Mra I represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.