It has been suggested that Escherichia coli lysine tRNA (E. coli tRNA(Lys)) has an unusual anticodon loop structure, judging from the abnormal CD pattern derived from 5-methylaminomethyl-2-thiouridine (mnm5s2U) located at the first position of the anticodon of the tRNA. Here, the structure of E. coli tRNA(Lys) was analysed by chemical modification and nuclear magnetic resonance (NMR) of the imino protons. The chemical probing approach showed that the anticodon arm region forms the ordinary stem-and-loop structure as expected from the standard clover-leaf structure, because N7 of G30, N3 of C40, N7 of A41 and N7 of A42 were protected under a native condition. By using the heteronuclear 15N-1H single quantum coherence (HSQC) spectra of 15N-labeled E.coli tRNA(Lys) and the analyses of the nuclear Overhauser effects (NOE) between the imino protons of non labeled E.coli tRNA(Lys), the imino protons in the acceptor stem was assigned, which indicates that the acceptor stem exists in an usual form. The tertiary base-base interactions was also detected. Thus, E.coli tRNA(Lys) seems to maintain a similar structure to the ordinary L-shaped tertiary structure, as a whole. The fine structure of the anticodon loop is now being analyzed.