We have studied the mechanism of action of the Serratia nuclease using deoxythymidine 3 ,5 -bis-(p-nitrophenyl-phosphate) as a substrate. A comparison of the activity with which the wild-type enzyme and several mutant enzymes attack this artificial substrate and herring sperm DNA, respectively, supports the suggestion that His 8 9 is the general base and a Mg 2 + ion bound to Glu 1 2 7 the general acid in the mechanism of phosphodiester bond hydrolysis by the Serratia nuclease, and that Asn 1 1 9 directly participates in catalysis, for example by transition state stabilisation. Arg 5 7 , Arg 8 7 and Arg 1 3 1 , essential for nuclease activity, are not needed for cleavage of the artificial substrate, suggesting that they are involved in binding and positioning of nucleic acid substrates.