A collection of highly nickel and cobalt-resistant enterobacteria were isolated from the Punta Gorda serpentine deposit (Moa, Cuba). The most nickel and cobalt resistant strain (termed C- 1) was assigned to Serratia marcescens by 16S rRNA analysis and DNA/DNA hybridization and the molecular mechanisms underlying its inducible cobalt and nickel resistance was investigated. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for Co- and Ni-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The three ORFs (ncrA, ncrB and ncrC) were cloned in E. coli. The predicted NcrA product was an NreB ortholog of the major facilitator protein superfamily and was central for Co/Ni resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in E. coli and caused decreased accumulation of Co and Ni in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the Ni–Co transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni and Co. As the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.