We have investigated the requirements for mature tRNA structure in the in vivo splicing of the Haloferax volcanii, intron-containing tRNATrp RNA. A partial tRNATrp gene, which contained only the anticodon stem-loop region of the mature tRNA, was fused to a carrier yeast tRNA gene for expression in H. volcanii. Transcripts from this hybrid gene were found to be processed by endonuclease and ligase at the tRNATrp exon-intron boundaries. These results verify that the substrate recognition properties of the halobacterial endonuclease observed in vitro reflect the properties of this enzyme in vivo, namely that mature tRNA structure is not essential for recognition by the endonuclease. The independence of these reactions on mature tRNA provides further support for a relationship between archaeal tRNA and rRNA intron-processing systems and highlight a difference in the substrate recognition properties between the archaeal and eucaryal processing systems. The significance of these differences is discussed in light of the observation that the tRNA endonucleases of these organisms are related.