The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5′ ends. A self-cleaving RNase P RNA–substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2′-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2′-hydroxyl that, when substituted with 7-deazaadenine or 2′-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate–phosphate in the tertiary structure of the ribozyme–substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2′-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.