Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50. With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected. The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus. The chromate resistance closely linked to the cnr locus was not affected by these mutations. In the Znr mutants, the resistance to zinc and nickel was constitutively expressed. Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain. Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system. A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt. The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc. Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype. Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.