The chrA gene of Pseudomonas aeruginosa plasmid pUM505 encodes the hydrophobic protein ChrA, which confers resistance to chromate by the energy-dependent efflux of chromate ions. Chromate-sensitive mutants were isolated by in vivo random mutagenesis. Transport experiments with cell suspensions of selected mutants showed that 51CrO4(2-) extrusion was drastically lowered as compared to suspensions of the strain with the wild-type plasmid, confirming that the mutations affected a chromate efflux system. DNA sequence analysis showed that most point mutations affected amino acids clustered in the N-terminal half of ChrA, altering either cytoplasmic regions or transmembrane segments, and replaced residues moderately to highly conserved in ChrA homologs. PhoA and LacZ translational fusions were used to confirm the membrane topology at the N-terminal half of the ChrA protein.