Several tRNAs in the hyperthermophilic bacterium Aquifex aeolicus are encoded in clusters and as part of ribosomal RNA operons, implying the requirement for tRNA processing by ribonuclease P (RNase P). Intriguingly, neither a gene for the RNA nor the protein component of this ubiquitous ribonucleoprotein enzyme has been hitherto identified in the sequenced genome of A. aeolicus, despite extensive data mining. As a result of the present study, primer extension analysis revealed that tRNAs in A. aeolicus possess canonical mature 5' ends; yet we were unable to demonstrate RNase P holoenzyme or RNase P RNA alone activity in A. aeolicus extracts under a variety of reaction conditions utilizing mono- and dimeric ptRNA substrates. Processing of dimeric ptRNA transcripts in extracts of A. aeolicus disclosed at least one endoribonuclease which cleaves in the A/U-rich spacer of the tandem ptRNA, reminiscent of bacterial RNase E-like enzymes.