In an attempt to identify the transcription initiation sites in chicken mitochondrial DNA, RNAs capped in vitro using vaccinia guanylyl transferase and [alpha-32P] GTP were analyzed. The most abundant labeled transcript was identified by RNA sequencing as the mitochondrial tRNA(His). Sequence analysis also revealed that this tRNA contains an extra guanylate residue at its 5' end, characteristic of the histidine tRNA family. The respective genomic region was also cloned and sequenced. In contrast to bacteria and the mitochondria of fungi and plants, the extra G of chicken mitochondrial tRNA(His) is not encoded in the gene. Therefore, the guanylate residue must be added post-transcriptionally, as demonstrated for the nuclear tRNA(His) in yeast and Drosophila. Analysis of a capped tRNA(His) precursor of chicken mitochondria suggests that addition of the extra G occurs independently of 3' end maturation. Since in the chicken mitochondrial tRNA(His) the extra G can be efficiently labeled by the capping assay, it should possess a 5'-terminal di- or triphosphate, which contrasts to the 5'-terminal monophosphate proposed for the nuclear encoded tRNA(His). Our results imply that the ability of a mitochondrial RNA to be capped in vitro does not necessarily prove that it contains a transcription initiation site.