Direct sequencing of human mitochondrial tRNALysshows the absence of editing and the occurrence of six modified nucleotides (m1A9, m2G10, Psi27, Psi28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNALys. Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m1A9 in this process.