Five mitochondrial (mt) tRNA genes from the filamentous fungus, Beauveria bassiana, were cloned and sequenced. The genes encoding the Val-, Ile-, Ser-, Trp- and Pro-accepting tRNAs were found clustered in the region 5′ to the lrRNA-encoding gene. The genes were 64–77% homologous with the equivalent genes from other filamentous fungi, 49–58% to yeasts with the exception of the Val-accepting tRNA-encoding gene which was 76%, and only slightly homologous with Escherichia coli. The B. bassiana mt genetic code was found to be similar to that of other fungal mitochondria in that the UGA codon is used as a signal for Trp rather than as a stop codon. Transcript analysis has revealed that the genes present in the tRNA cluster are transcribed and processed into tRNA-size products. Secondary structure models proposed for the gene products show that conservation of tRNA secondary structure also exists. The presence of a GGC sequence rather than a GGU sequence in the D-loop of the tRNA Trp-encoding gene is a feature unique to the B. bassiana mt tRNA. An unconventional G-A base pair present in the D-stem of the tRNA Ser-encoding gene is a feature conserved in the mt tRNA of other filamentous fungi. Comparison of the B. bassiana tRNA-encoding genes with those of two other filamentous fungi and two yeasts revealed that the differences between closely related species favoured transition-type mutations.