DNA sequencing was used to confirm Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in endocervical swab samples. DNA in residues of the samples with positive results by 2 commercial kits was subjected to nested polymerase chain reaction (PCR) amplification. The nested PCR amplicons were used as templates for direct automated DNA sequencing. A 40-base signature sequence was sufficient to achieve unequivocal validation of C trachomatis cryptic plasmid and gonococcal opa gene DNA. DNA with a signature sequence specific for C trachomatis was identified in all 14 samples and for N gonorrhoeae in all 13 samples with positive results by the commercial kits for these 2 microbes. In a low-prevalence population, PCR retesting of 289 samples with initial negative results by a non-nucleic acid amplification assay revealed 3 samples positive for C trachomatis and 2 samples positive for N gonorrhoeae that were missed by the commercial kit. DNA sequencing is a useful tool in validating molecular diagnostics.