Chlorate-resistant mutants with none of the usual pleiotropic effects such as defective nitrate reductase activity were isolated fromEscherichia coliK-12. These chlorate-resistant mutants (designatedchlHW) did not yield strains with a high level of nitrofurantoin resistance following selection with nitrofurantoin. The chlorate-resistance mutation reduced the nitrofurantoin resistance of high-level mutants to an intermediate level. Further mutation to resistance to streptomycin and other aminoglycoside antibiotics suppressed the effect ofchlHWon the level of nitrofurantoin resistance. Other chlorate-resistance genes examined did not have the same effect on nitrofurantoin resistance aschlHW. The gene was cotransducible (P1) with intermediate-level nitrofurantoin resistance andproC. It is suggested that thechlHWmutation may enhance the accumulation of nitrofurantoin inside the cell since it is known that a multiple aminoglycoside-resistance mutation with pleiotropic effects on the cell membrane can also confer high-level resistance to nitrofurantoin.