pVS438, a clone of part of the Tra 2 region of RP1 in RSF1010, confers two unusual phenotypes: poor growth (Slo+) in Pseudomonas aeruginosa PAO and surface exclusion (Sfx+) in Escherichia coli K12. Both of these phenotypes were found to be encoded by a 1.8-kb fragment of RP1 (from 25.9-27.7 kb) that spans the traB gene. However, whether both phenotypes, neither, or only Slo+ is expressed by this fragment depends on its location and orientation in RSF1010. In pVS438, where this fragment occurs in the SmR locus of RSF1010, expression of the Sfx+ phenotype is due to augmented transcription from the two promoters that cotranscribe the SuRSmR genes. When augmentation is abolished by insertion of Tn5 between these promoters and the cloned fragment, or by insertion of the fragment elsewhere in RSF1010, a Slo+Sfx- phenotype results. DNA that confers only the Slo+ phenotype was mapped to the 26.2-26.8 kb region of RP1 between traE and traB and the designation, traS, given to the gene responsible. Despite the recognition of a traS+ (Slo+) component of DNA within that encoding the Slo+ and Sfx+ phenotypes, this gene seems nevertheless to be responsible for the Sfx+ phenotype since hydroxylamine-induced Slo- mutants of pVS438 are usually also Sfx-. These apparently conflicting observations and the precise interplay between the Slo+, Sfx+, and TraB+ phenotypes were not resolved. Finally, traS is not essential for plasmid transfer since pVS438 and a Slo-Sfx- derivative of it can both equally complement an RP1tra-deletion mutant of part of the Tra 2 region.