Computational studies predict the simultaneous presence of two and even three introns in certain crenarchaeal tRNA genes. In these multiple-intron-containing pretRNAs, the introns are nested one inside the other and the pretRNA folds into a conformation that is anticipated to allow splicing of the last intron only after splicing the others. A set of operations, each consisting of two cleavages and one ligation, therefore needs to be carried out sequentially. PretRNAs containing multiple introns are predicted to fold, forming bulge-helix-bulge (BHB) and BHB-like motifs. The tRNA splicing endonuclease should recognize these motifs. To test this hypothetical scenario, we used the homotetrameric enzyme from Methanocaldococcus jannaschii (METJA) and the heterotetrameric enzyme from Sulfolobus solfataricus (SULSO). On the basis of our previous studies, the METJA enzyme should cleave only the BHB structure motif, while the SULSO enzyme can in addition cleave variant substrate structures, like the bulge-helix-loop (BHL). We show here that the processing of multiple-intron-containing pretRNA can be observed in vitro.