Transition metals are known to cause toxic effects through their interaction with oxygen, but toxicity under anoxic conditions is poorly understood. Here we investigated the effects of iron (Fe) and copper (Cu) on the anaerobic growth and gene expression of the purple phototrophic bacterium Rhodopseudomonas palustris TIE-1. We found that Fe(II) and Cu(II) act synergistically to delay anaerobic growth at environmentally relevant metal concentrations. Cu(I) and Cu(II) had similar effects both alone and in the presence of ascorbate, a Cu(II) reductant, indicating that reduction of Cu(II) to Cu(I) by Fe(II) is not sufficient to explain the growth inhibition. Addition of Cu(II) increased the toxicity of Co(II) and Ni(II); in contrast, Ni(II) toxicity was diminished in the presence of Fe(II). The synergistic anaerobic toxicity of Fe(II) and Cu(II) was also observed for Escherichia coli MG1655, Shewanella oneidensis MR-1, and Rhodobacter capsulatus SB1003. Gene expression analyses for R. palustris identified three regulatory genes that respond to Cu(II) and not to Fe(II): homologs of cueR and cusR, two known proteobacterial copper homeostasis regulators, and csoR, a copper regulator recently identified in Mycobacterium tuberculosis. Two P-type ATPase efflux pumps, along with an F(o)F(1) ATP synthase, were also upregulated by Cu(II) but not by Fe(II). An Escherichia coli mutant deficient in copA, cus, and cueO showed a smaller synergistic effect, indicating that iron might interfere with one or more of the copper homeostasis systems. Our results suggest that interactive effects of transition metals on microbial physiology may be widespread under anoxic conditions, although the molecular mechanisms remain to be more fully elucidated.