Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it has been shown previously that there are nonluminous vibrios that possess lux genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus Vibrio. Screening of over 600 Vibrio isolates with a luxA gene probe revealed that approximately 5% carried the luxA gene. Bioluminescence assays of the luxA-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions. Strain BCB451 has an IS10 insertion sequence in luxA, a mutated luxE stop codon, and a truncated luxH. Strain BCB494 has a 396-bp deletion in luxC, and strain BCB440 has a frameshift in luxC. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete lux operons in natural isolates.