Queuosine (Q), one of the most complex modifications occurring at the wobble position of tRNAs with GUN anticodons, is implicated in a number of biological activities, including accuracy of decoding, virulence, and cellular differentiation. Despite these important implications, its biosynthetic pathway has remained unresolved. Earlier, we observed that a naturally occurring strain of Escherichia coli B105 lacked Q modification in the tRNAs. In the present study, we developed a genetic screen to map the defect in E. coli B105 to a single gene, queC (renamed from ybaX), predicted to code for a 231-amino-acid-long protein with a pI of 5.6. As analyzed by mobility of tRNA(Tyr) on acid urea gels and two-dimensional thin-layer chromatography of the modified nucleosides, expression of QueC from a plasmid-borne copy confers a Q+ phenotype to E. coli B105. Further, analyses of tRNA(Tyr) from E. coli JE10651 (queA mutant), its derivative generated by deletion of chromosomal queC (queA deltaqueC), and E. coli JE7325, deficient in converting preQ0 to preQ1, have provided the first genetic evidence for the involvement of QueC at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. In addition, we discuss the possibilities of collaboration of QueC with other cellular proteins in the production of preQ0.