The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precursor tRNAs and catalyzes the removal of 5' leader nucleotides. Biochemical and photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate complex. To investigate the local three-dimensional environment of the 5' leader, we employed the short-range photo-cross-linking agent 4-thiouridine (s(4)U). The s(4)U photoagent was incorporated into a series of pre-tRNA substrates containing unique uridine residues in the 5' leader sequence at positions -1, -3, -5, -7, or -10. The modified substrates formed high-affinity complexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis. Locations of the cross-linked nucleotides in the ribozyme and pre-tRNA were determined by reverse transcriptase primer extension. Photoagents incorporated into the 5' leader detected discrete elements of ribozyme structure in a progression from J18/2 to L15 to P3. Importantly, all of the cross-linked species retained the ability to cleave the covalently attached pre-tRNA, indicating that the cross-links reflect the native structure of the ribozyme-substrate complex. Together with available structural and biochemical data, the cross-linking results suggest a model for the position of the 5' leader within the ground-state ribozyme-substrate complex.