Antigenic variation of gonococcal pilin involves a family of variable genes that undergo homologous recombination, resulting in transfer of variant sequences from the pilS silent gene copies into the complete pilE expression locus. Little is known about the specific recombination events that are involved in assembling new variant pilin genes in vivo. One approach to understanding pilin variation in vivo is to carry out experimental human infections with a gonococcal strain having a fully characterized repertoire of pilin genes, so that the specific recombination events occurring in vivo can be determined. To this end, the authors cloned, sequenced and mapped the pilin genes of strain FA1090 of Neisseria gonorrhoeae. This strain contains one pilE locus and 19 silent gene copies that are arranged in five pilS loci; the pilE locus and four of the pilS loci are clustered in a 35 kb region of the chromosome. The general features of the pilin loci in FA1090 are similar to those in strain MS11, in which the mechanism of pilin variation has been extensively studied. However, none of the silent copy sequences are identical in the two strains, which emphasizes the extreme variability in this gene family among gonococci. Three male volunteers were inoculated with the same variant of strain FA1090 and developed urethritis within 2--4 d. The pilE gene sequences from a total of 23 colonies cultured from the subjects were analysed, determining which pilS silent copy donated each portion of the expressed pilE genes. There were 12 different pilin variants, one of which was the original inoculum variant, among the in vivo-expressed pilE gene sequences. The pilE of the inoculum variant was derived entirely from a single silent copy (pilS6c1). However, the pilE genes in the majority of the colonies cultured from the infected subjects were chimeras of sequence derived from two or three silent copies. Recombination to generate new pilE sequences involved exchange of single variable minicassettes, multiple minicassettes, entire silent gene copies, or (rarely) recombination within a minicassette.