Nickel and cobalt are essential micronutrients for many microorganisms and serve as enzyme cofactors that catalyze a diverse array of reactions. One complication is that high concentrations of these transition metal ions are toxic to cells, leading some prokaryotes to evolve sophisticated homeostatic mechanisms to regulate their transmembrane uptake or efflux. The biosynthesis of nickel and cobalt metalloenzymes requires the intracellular allocation of the metals to the appropriate apoproteins, often in an intricate process that involves the cooperative activity of accessory proteins. Here, we highlight the molecular physiology of nickel and cobalt cation metabolism in Escherichia coli and summarize additional nickel- or cobalt-dependent processes and homeostatic mechanisms found in other microorganisms.