Copper is an essential cofactor for many enzymes, and at over a threshold level, it is toxic for all organisms. To understand the mechanisms underlying copper homeostasis of the gram-positive bacterium Bacillus subtilis, we have performed microarray studies under copper-limiting conditions. These studies revealed that the ycnJ gene encodes a protein that plays an important role in copper metabolism, as it shows a significant, eightfold upregulation under copper-limiting conditions and its disruption causes a growth-defective phenotype under copper deprivation as well as a reduced intracellular content of copper. Native gel shift experiments with the periplasmic N-terminal domain of the YcnJ membrane protein (135 residues) disclosed its strong affinity to Cu(II) ions in vitro. Inspection of the upstream sequence of ycnJ revealed that the ycnK gene encodes a putative transcriptional regulator, whose deletion caused an elevated expression of ycnJ, especially under conditions of copper excess. Further studies demonstrated that the recently identified copper efflux regulator CsoR also is involved in the regulation of ycnJ expression, leading to a new model for copper homeostasis in B. subtilis.