Most recommended restriction buffen contain Na and d". However, in bacteria the most abundsnt intracelhilar cation and anion are usually potassium and gM îruK*. respectively (1). Furthermore, restriction endoaocleases cleave DNA in potassium glutamate (KGbj) over a much broader concentration range than they do in N»Q (2). These facts encouraged as to investigate the possibility that we could use KGlu in a chloride-free buffer and achieve normal level] of activity for all restriction endonuclease*. We have tested fifty-five restriction endonucleases for their ability to cleave DNA in a series of KGlu buffers (KGB, see Table 1) and compared the level of activity with that found under conditions recommended by the vendors (New England BioJabs, Boehringer Mannheim Biochem. and International Biotech. Inc.). Assays were performed u partial digests (0.2 units per ng of DNA in 30 ul for 30 min.) and as overnjfht digestions with excess enzyme to ensure that no lost of specificity (star activity) occurred. Most restriction endonudeases, potymerases and Ugase showed broad KGta concentration optima and aD enzymes functioned in 100 raM KGlu (IX KGB). Reducing agent was not normally required Some enzymes worked well in concentrations of KGlu over 400 mM (data not presented). KGB can be used to simplify laboratory prccedms tnrtnrting double digesa, DNA cleavage followed by end-labeling, or me digestion of DNA embedded in agarose prior to pulsed field gel electrophoresw DNA in KGB can be phenol extracted and ethanol precipitated using sandard protocols.