The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.