The Neisseria gonorrhoeae pilE gene codes for a type IV pilin, the major subunit of pili which constitute an essential virulence factor during gonococcal infection. Expression of pilE seems to be highly regulated, which may allow piliation to adapt to growth conditions. From an N. gonorrhoeae genomic library, we selected plasmid pNG200 encoding a protein (RegF) which caused a 5-fold increase in the expression of pilE::cat fusion in Escherichia coli. This regulation was mediated via the complex pilE promoter region, comprising potential sigma 70- and sigma 54-dependent promoters, and could not be observed in the absence of an active sigma 54 factor. The RegF protein (23,149 Da) showed 42% identity with the E. coli "stringent starvation protein", SspA. This protein was shown to interact with the RNA polymerase holoenzyme and to play a role in the expression of at least 11 proteins in E. coli. In an N. gonorrhoeae strain carrying a regF::mTn3Cm3 mutation constructed by allelic exchange, it was observed that pilin expression was enhanced. Our results were consistent with a model in which (i) in N. gonorrhoeae, RegF acts as a negative regulator of pilE transcription, and (ii) in E. coli, RegF increases pilE transcription by preventing sigma 54-associated steric hindrance at pilE promoters described previously.