N(6)-threonylcarbamoyladenosine (t(6)A) is a universally conserved, essential modified nucleoside found in transfer RNAs (tRNAs) responsible for ANN codons in all three domains of life. t(6)A has a crucial role in maintaining decoding accuracy during protein synthesis. The presence of t(6)A in cellular tRNAs has been well documented for more than four decades. However, under conditions optimized for nucleoside preparation, we detected little t(6)A in tRNAs from Escherichia coli. Instead, we identified a new modified base named 'cyclic t(6)A' (ct(6)A), which is a cyclized active ester with an oxazolone ring. An E1-like enzyme, CsdL (renamed as TcdA), which catalyzes ATP-dependent dehydration of t(6)A to form ct(6)A, was also identified. Two yeast homologs of tcdA, YHR003C (TCD1) and YKL027W (TCD2), were required for ct(6)A formation and respiratory cell growth. ct(6)A was involved in promoting decoding efficiency. Structural modeling suggests that ct(6)A recognizes the first adenine base of ANN codon at the ribosomal A site.