Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre-pilin peptidase, were unable to process the pre-pilin subunit into pilin and thus were non-piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S-pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide-binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.