The transcriptional organization of the Tra2 region controlling conjugation by the narrow host range plasmid R91–5 of Pseudomonas aeruginosa has been determined. This region of 15·2 kilobase pairs of DNA encodes ten cistrons and specifies the synthesis and functionality of sex pili. The order of the cistrons was previously shown to be traW, (S, Z), U, (V, R), Q, T, Y, X (those within parentheses could not be ordered with respect to each other). The organization was determined by complementation tests for the restoration of plasmid transfer between transposon Tn7 or Tn501 induced insertion mutants and point mutants representative of the ten cistrons. Polarity mutations induced by Tn7 permitted the identification of three operons. Cistrons traW, Z, S, U, V, R and Q formed a large operon with transcription postulated to be in that order. The second consisted of tra T and tra Y also in that order and the third consisted of only one cistron, traX. Polarity was not observed with Tn501 insertions as they still permitted full expression of cistrons distal to the site of insertion with respect to the proposed promoter. Despite the unexpected behaviour of this transposon it nevertheless led to the identification of two previously unidentified cistrons, traO and traP being between traZ and S and traR and V, respectively.