The activities of 17 endonucleases: the restriction endonucleases AvaI, BamHI, EcoRI, HindII1, PstT and SalI, which cleave pBR322 DNA once: AluI, AvaII, CfnI, HaeIII, HhaI, HinfI, HpaII and TuqI, which cut pBR322 D N A several times, and three 'unspecific' nucleases (S1 nuclease, staphylococcal nuclease and DNase I from bovine pancreas) were determined between 0 and 65 '-C. The reaction was followed by the disappearance of covalently closed circular pBR322 DNA, using the alkaline ethidium fluorescence assay of Morgan et al. [Nucleic Acids Res. (1979) 7, 547-5941; the activity of T4 DNA ligase was similarly measured by the conversion of nicked circular D N A to closed circular DNA. For each enzyme, small aliquots of the same solution were incubated at different temperatures simultaneously in a temperature gradient device, resulting in a high relative precision.The experimental results are summarized by the simplest possible theoretical description, using linear or exponential kinetics and apparent activation energies E, for the enzymatic reaction, E i for the enzyme inactivation and Ti for the inactivation temperature. To a good approximation these three parameters suffice for describing the temperature dependence of the activity of most of the enzymes.The recent increase in knowledge about the chemical structure of D N A would not have been possible without the discovery and the purification of many enzymes which modify DNA. (An excellent introduction of the properties of such enzymes is found in a book by Kornberg [l].)Restriction endonucleases from many different organisms recognize particular sequences in double-helical DNA, cleaving it at precisely defined positions (e.g. [2-51). Besides their indispensible role in recombinant D N A research, they provide a unique possibility for studying DNA-protein interactions. DNA ligase is able to reverse the action of such nucleases, allowing new recombinations of DNA in vitro [6,7].The activity of these enzymes is usually measured by assays which involve electrophoresis and/or the use of radioactively labeled compounds. The fluorescence increase due to the binding of ethidium to D N A provides a rapid, sensitive and economic way for measuring the activity of a large number of DNA-modifying enzymes and has recently been reviewed by Morgan et al. [8,9]. Measuring the fluorescence in alkaline solution before and after heating allows the accurate determination of the fraction of covalently closed circular DNA in a solution. Using this DNA from the plasmid pBR322 [lo] as substrate for endonucleases and measuring its degradation by the ethidium fluorescence assay provides a fast and quantitative method for evaluating, for example, the activity of restriction endonucleases.