The pilus of Neisseria gonorrhoeae (the gonococcus Gc), the causative agent of gonorrhoea, promotes attachment of the gonococcus to the host epithelium and is essential for the establishment of disease. The ability of N. gonorrhoeae to infect previously exposed individuals is partially due to pilus antigenic variation. In addition, variation of the pilus has been proposed to function in the adaptation of the gonococcus to host environments. Previously, we described the development of a competitive reverse transcriptase (RT)-PCR assay that quantifies the frequency of pilin antigenic variation within a gonococcal population. Using this assay, the effect of different biologically relevant environmental conditions on the frequency of pilin antigenic variation was tested. Of the environmental conditions examined in vitro, only limited iron affected a significant change in the frequency of antigenic variation. Further investigation revealed that an observed increase in pilin antigenic variation reflected an increase in other DNA recombination and DNA repair processes within iron-starved cultures. In addition, this low iron-induced increase was determined to be independent of changes in RecA expression and was observed in a Fur mutant strain. As gonococci encounter conditions of low iron during infection, these data suggest that iron-limitation signals for increased recombinational events that are important for gonococcal pathogenesis.