Analysis of RNA isolated from Pseudomonas aeruginosa PA103 and PAKS grown under Fe2+-limiting (0.08 microgram/ml) and Fe2+-sufficient (10 micrograms/ml) conditions demonstrated that exotoxin A (ETA) expression is regulated by Fe2+ at the level of transcription. S1 nuclease mapping revealed two 5' termini of the tox transcript, 89 base pairs (bp) (S1A) and 62 bp (S1B) 5' to the ETA initiation codon. There appeared to be no consensus promoter sequence for either tox transcript. An 8-bp direct repeat was found 5' to the start of transcript S1A. Transcript S1B mapped 8 bp upstream of a dodecamer sequence conserved between the ETA and phospholipase C genes of P. aeruginosa. Multicopy plasmids in which the expression of ETA is directed from the Escherichia coli trp promoter (ptrpETA-RSF1010) or the tox promoter (pCMtox) were constructed and mobilized into a Tox-P. aeruginosa strain, WR5. WR5 synthesized and secreted high levels of ETA when it was expressed from the E. coli trp promoter; however, the synthesis of ETA from its own promoter in this strain was very low. These and other data suggest that the expression of ETA is under a positive control mechanism. A fusion of the ETA promoter fragment to lacZ was constructed. Use of this fusion plasmid revealed that this DNA fragment directed the synthesis of beta-galactosidase in E. coli at very low levels and that the synthesis of beta-galactosidase from this fusion in E. coli was not regulated by Fe2+.