In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes. Protein components from the Tetrahymena thermophila and human RNase P holoenzymes form specific products in photoreactions containing [4-thio]-uridine-labeled pre-tRNAGln. The HeLa RNase P RNA in neither the presence nor the absence of holoenzyme protein components formed cross-link products to the pre-tRNAGln probe. Parallel photo-cross-linking experiments with the Escherichia coli RNase P holoenzyme revealed that only the bacterial RNase P RNA forms specific substrate photoadducts. A protein-rich active site for the eukaryotic RNase P represents one unique feature that distinguishes holoenzyme organization between bacteria and eukaryotes.