RNase T was first identified as an enzyme responsible for end turnover of tRNA in Escherichia coli. Its activity, specific for tRNA-C-C-A, catalyzes the release of tRNA-C-C and AMP. RNase T, along with several other RNases, plays a role in maturation of several other RNA species by a similar limited nuclease activity. In previous work, we identified the gene for RNase T, rnt, as a high copy suppressor of the UV sensitivity conferred by deficiency in three single-strand DNA-specific exonucleases, RecJ, exonuclease I, and exonuclease VII. This suggested that RNase T may process DNA substrates as well. In this work, we show that purified RNase T possesses a potent 3' to 5' single-strand DNA-specific exonucleolytic activity. Its Km for single-strand DNA substrates is many orders of magnitude lower than that for tRNA, suggesting that single-strand DNA may be a natural biological substrate for RNase T. We suggest that the DNase activity of RNase T may play a role in end trimming reactions during DNA recombination and/or DNA repair.