Membrane preparations from a series of Hfr mutant strains of Escherichia coli K12 deleted in the promoter distal end of the F transfer operon were analyzed. Deletions which extended into traG, as expected, had no discernible effect on synthesis of membrane F-pilin. A more extensive deletion in strain K1777 which eliminated traH activity similarly had no effect on F-pilin synthesis. Membranes from three other TraF+ TraH- deletion strains, as well as membranes from all strains carrying deletions extending into traF or further, lacked F-pilin, however. Since traH amber mutations do not affect synthesis of membrane pilin (Moore et al. 1981 b) we conclude that a gene required for F-pilin biosynthesis is located between traF and traH. We have named this gene traQ. Further evidence for traQ and an assay for its activity was obtained by examining the products of a TraM+ TraJ+ TraA+ lambda transducing phage, KI lambda 13, in UV irradiated cells. Infection of F- cells with KI lambda 13 does not result in F-pilin synthesis. Membrane pilin is synthesized as a product of the transducing phage if an Flac or Hfr irradiated host is used, however. Mutant analysis demonstrated that this synthesis is independent of host expression of traA, traL, traE, traK, traB, traV, traW, traC, traU, traF, or traH, but dependent on expression of the traF-traH region. We interpret our data to indicate that an activity encoded by traQ is required for the conversion of traA product to F-pilin.