Many illnesses and infections are exacerbated and/or caused by biofilms. Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, which can lead to persistent infection. Persistent infection can result in pelvic inflammatory disease, tubo-ovarian abscesses, infertility, and ectopic pregnancy. N. gonorrhoeae has been shown to form biofilms over glass, primary and immortalized cervical cells, and during natural cervical infection. Asymptomatic infection occurs in only 1% of infected males, and the infection site is subject to periodic rapid fluid flow, which may limit biofilm formation. Thus, biofilm formation may specifically play an important role in the infection of women and could contribute to the infrequent occurrence of symptoms. Prior to work presented in this dissertation, little was known about biofilm formation by N. gonorrhoeae. Therefore, we elected to compare the transcriptional profiles of biofilms to their planktonic counterparts, to identify genetic pathways involved in biofilm formation and maintenance. We found that 3.8% of the genome was differentially regulated, and that genes involved in anaerobic metabolism and oxidative stress tolerance were up-regulated in biofilm, while genes involved in aerobic metabolism were down-regulated. We determined that expression of aniA , ccp, and norB is required for robust biofilm formation over glass and human cervical cells, and anaerobic respiration occurs in the substratum of gonococcal biofilms. Disruption of the norB gene resulted in severe attenuation of biofilm formation. We determined that the accumulation of nitric oxide (NO) contributes to the phenotype of a norB mutant and can retard biofilm formation when present at sublethal concentrations. However, higher concentrations of NO can enhance biofilm formation in the absence of nitrite. NO enhances biofilm formation in an aniA mutant, but cannot completely restore biofilm formation, suggesting that NO can support anaerobic growth, although nitrite is preferred. We determined that the majority of the genes involved in gonococcal oxidative stress tolerance are required for normal biofilm formation, as mutations in the following genes resulted in biofilm attenuation over cervical cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm formation may represent an adaptation for coping with the stresses present in the female genitourinary tract.