Pseudomonas aeruginosa binds to eukaryotic cells via both pilus and nonpilus adhesins, while binding of P. aeruginosa to mucin is pilus independent. To characterize genes involved in non-pilus-mediated adherence, transposon mutants of the nonpiliated strain P. aeruginosa PAK-NP that are unable to bind to cells or mucins were isolated. Two such mutants, P. aeruginosa B164 and P. aeruginosa RR18, were identified previously as deficient in binding to eukaryotic cells or mucins as well as nonmotile. The transposon insertion in each of these strains was mapped to the same gene. Sequence analysis of both DNA flanking the transposons and plasmids that could complement the mutations indicated that this open reading frame encodes a putative protein homolog of both Escherichia coli FliO and Erwinia carotovora subsp. atroseptica MopB. The transposons in both of these mutants are nonpolar, since the addition of the P. aeruginosa fliO gene in trans restored adherence to both cells and mucins to these mutants. The cloned fliO gene also complemented the motility defect of both B164 and RR18. A 1.6-kb KpnI fragment from the PAK chromosome that contained the fliO gene was sequenced. The fliO gene appears to be part of an operon with a complete open reading frame upstream of the FliO homolog encoding a putative protein homolog of FliN of both E. coli and Salmonella typhimurium. The partial open reading frame downstream of fliO encodes a putative homolog of both E. coli and S. typhimurium FliP. The fliN gene is flanked on its 5'-end by the 3'-end of a homolog of a fliM gene. The P. aeruginosa FliN protein was identified with a T7 expression system, while all attempts to identify the P. aeruginosa FliO protein were unsuccessful. Homologs of P. aeruginosa FliO are involved in the biosynthesis of flagella, but the function of FliO in this biosynthetic process remains unknown. Further study should reveal the precise role of P. aeruginosa FliO in non-pilus-mediated adherence, which could include regulation of expression or localization of a nonpilus adhesin.