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We have developed a sensitive continuous assay for nucleases using proton release. The assay has been applied to the determination of the kinetics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of Kcat/K(m) on sequence and structure of short oligonucleotide substrates has been measured: increasing lengths of AnTn sequences decrease the rate of cleavage. G.A mismatches in which the bases pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem G.A mismatches which use amino pairing and have BII phosphodiesters, are refractory to DNase I. Also, the DNA strands of DNA.RNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove-binding ligand propamidine. A value of 3 microM has been determined for binding to the sequence d(CGCGAATTCGCG)2, showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.