An in vitro selection endeavor previously executed by our laboratory led to the isolation of a set of RNA-cleaving deoxyribozymes that thrive under acidic conditions [Liu, Z., Mei, S. H., Brennan, J. D., and Li, Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One of these sequences, coined pH5DZ1, is a 100-nucleotide (nt) cis-acting enzyme that was found to exhibit high cleavage activity near pH 5. Herein, we seek to deduce the properties and sequence requirements of this enzyme. This deoxyribozyme was found to cleave a 23-nt chimeric DNA-RNA substrate, which contains a single ribonucleotide flanked by fluorophore- and quencher-modified nucleotides on each side of the cleavage junction. Extensive nucleotide deletion experiments indicated that only 42 bases within the original enzyme sequence are required for catalysis. Results from a reselection experiment further revealed that 26 of these nucleotides are absolutely conserved. In addition to sequence analysis and minimization studies, we successfully designed a trans-acting variant of this enzyme. Characterization of the cleavage products produced upon pH5DZ1-mediated RNA cleavage and analyses of possible structures of pH5DZ1 provided us with insights into the catalytic mechanism of pH5DZ1 and characteristics of deoxyribozymes that retain their activity under acidic conditions.