Nucleotide sequence of the riboflavin synthesis genes ribE, ribB, ribH, ribA (GenBank Accession No. AF364106) resided in the lux operon of Photobacterium leiognathi PL741 has been determined, and the amino acid sequences of riboflavin synthetase (RibE), DHBP synthetase (RibB), lumazine synthetase (RibH), GTP cyclohydrolase II (RibA) encoded by the riboflavin synthesis genes are deduced. Nucleotide sequence reveals that the ribE gene encodes the riboflavin synthetase responsible for converting lumazine to riboflavin, the ribB gene encodes the DHBP synthetase responsible for 3,4-dihydroxyl-2-butanone 4-phosphate synthesis, the ribH gene encodes the lumazine synthetase responsible for lumazine synthesis, and the ribA gene encodes the GTP cyclohydrolase II responsible for lumazine synthesis. Functional analysis illustrates that the specific segments lay behind the ribH and ribA genes might form potential loops Omega (oT) and Omega (TI)-Omega (TII); Omega (oT) is functioned as mRNA stability loop or/and for subregulation by alternative modulation, and Omega (TI)-Omega (TII) could be the transcriptional terminator of the lux operon. The gene order of the ribE, ribB, ribH, ribA genes resided in the lux operon and linked to the lum operon is <--ter(o)-lumQ-lumP-R&R-luxC-luxD-luxA-luxB-luxN-luxE-luxG-ribE-ribB-ribH-ribA-ter-->, (R&R: regulatory region; ter: transcriptional terminator), whereas the R&R is the regulatory region for the lum and the lux operons, and ter and ter(o) are the transcriptional terminators for the lux and lum operons.