The Escherichia coli F plasmid gene, traG, is required for two stages of the conjugation process: pilus biosynthesis and mating aggregate stabilization. The nucleotide sequence of traG has been determined and the topology of its product in the cytoplasmic membrane analysed using protease accessibility experiments. Complementation analysis employing plasmid deletions revealed a correlation between an N-terminal periplasmic segment of the protein product (TraGp) and its pilus assembly activity. Production of an anti-TraGp antiserum has facilitated the detection of TraGp*, a possible internal cleavage product of TraGp. Although its function is unknown. TraGp* is located in the periplasm and has been shown to possess sequences required for aggregate stabilization. The detection of TraGp* raises the possibility that the two functions of traG are carried out by separate products.