Using transposon Tn5 inactivation technology a collection of Escherichia coli mutants defective in riboflavine biosynthesis was obtained. All mutations were distributed within three linkage groups. With the help of P1-transduction mapping, group I mutations (ribA locus) were localized near cysB locus (28 min of the standard 100 min E. coli map) and mutations of group II (ribB locus) were mapped near tolC locus (66 min). The location of group III mutations was approximately determined by the F' complementation analysis: this linkage group lies in the region of 56-60 min of the E. coli map.