A gene cluster composed of nine open reading frames (ORFs) involved in Ni(2+), Co(2+), and Zn(2+) sensing and tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 has been identified. The cluster includes an Ni(2+) response operon and a Co(2+) response system, as well as a Zn(2+) response system previously described. Expression of the Ni(2+) response operon (nrs) was induced in the presence of Ni(2+) and Co(2+). Reduced Ni(2+) tolerance was observed following disruption of two ORFs of the operon (nrsA and nrsD). We also show that the nrsD gene encodes a putative Ni(2+) permease whose carboxy-terminal region is a metal binding domain. The Co(2+) response system is composed of two divergently transcribed genes, corR and corT, mutants of which showed decreased Co(2+) tolerance. Additionally, corR mutants showed an absence of Co(2+)-dependent induction of corT, indicating that CorR is a transcriptional activator of corT. To our knowledge, CorR is the first Co(2+)-sensing transcription factor described. Our data suggest that this region of the Synechocystis sp. strain PCC 6803 genome is involved in sensing and homeostasis of Ni(2+), Co(2+), and Zn(2+).