RNase D, a putative tRNa processing nuclease, has been purified about 1,000-fold from extracts of Escherichia coli to apparent homogeneity, as judged by acrylamide gel electrophoresis under nondenaturing and denaturing conditions and by gel electrofocusing. The purified enzyme is a single chain protein with a molecular weight of 40,000 and an isoelectric point of about 6.2. Spectral analysis indicated that RNase D is devoid of nucleic acid. Amino acid analysis suggested a low content of cysteine, and this was confirmed by the relative insensitivity of the enzyme to sulfhydryl group reagents. RNase D is sensitive to inactivation by elevated temperatures but can be protected by a variety of RNAs, including those which are not substrates for hydrolysis. The relation of RNase D to other known E. coli ribonucleases and to other previously identified processing activities, is discussed.