The endoribonuclease RNase P processes tRNA-like structures that are assembled out of two separate strands. In these bimolecular constructs, one of the strands is cleaved by the enzyme, and the other one is called the external guide sequence (EGS). A number of EGS with different mutations and deletions were tested for the ability to induce cleavage with human RNase P. Different domains of the original tRNAtyr-like structure were deleted or modified. The anticodon stem and loop and the variable loop could be deleted without a detrimental effect on recognition by RNase P. Modifications in the lengths of T stem and aminoacyl acceptor stem led to a decrease in the relative amount of cleavage, whereas modifications of the D stem were more permissible. Single nucleotide deletions in the T loop reduced cleavage to different extents, depending on the position. Values for the Kd of complex formation of bimolecular constructs with annealing arms of varying lengths ranged from 0.2 nM to 28 nM. A cleavage rate of 1 min(-1) was measured for both the bimolecular target-EGS complex and tRNA precursor.