Ribonuclease P (RNase P) catalyzes the 5'end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions [True, H. L., and Celander, D. W. (1998) J. Biol. Chem. 273, 7193-7196]. We now report that Pop3p, an essential protein subunit of the holoenzyme in Saccharomyces cerevisiae, displays novel RNA-binding properties. A recombinant form of Pop3p (H6Pop3p) displays a 3-fold greater affinity for binding pre-tRNA substrates relative to tRNA products. The recognition sequence for the H6Pop3p-substrate interaction in vitro was mapped to a 39-nucleotide long sequence that extends from position -21 to +18 surrounding the natural processing site in pre-tRNA substrates. H6Pop3p binds a variety of RNA molecules with high affinity (Kd = 16-25 nM) and displays a preference for single-stranded RNAs. Removal or modification of basic C-terminal residues attenuates the RNA-binding properties displayed by the protein specifically for a pre-tRNA substrate. These studies support the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.