DL-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu2+ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu2+ but not of excessive Zn2+ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.