The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni²⁺ transport assay was used to monitor uptake and accumulation of ⁶³Ni²⁺ into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on ⁶³Ni²⁺ transport and accumulation. Cells grown either in the presence or absence of CO transported Ni²⁺ with a K_m of 19 ± 4 µM and a V_max of 310 ± 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni²⁺ the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn²⁺, Mg²⁺, Ca²⁺, and Zn²⁺ showed no inhibition of ⁶³Ni²⁺ transport but Co²⁺, Cd²⁺, and Cu²⁺ inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4°C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N,N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni²⁺ transport. ⁶³Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated ⁶³Ni²⁺ accumulation coincided with the appearance of CODH activity in the culture, suggesting that the ⁶³Ni²⁺ was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased ⁶³Ni²⁺ accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated ⁶³Ni²⁺ like cells unexposed to CO.