Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.