The gram-negative bacteria Escherichia coli (E. coli) is a very useful prokaryotic model for testing the toxicity of ZnO nanoparticles (nano-ZnO). This toxicity is often linked to Zn2+ released from nanoparticles in the culture medium, and nano-ZnO dissolution in different media is clearly established. Here, two model E. coli strains MG1655 and W3110 both descendant from the original K-12 showing slight differences in their genome were submitted to nano-ZnO or Zn2+ in order 1> to refine the nano-ZnO toxicity mechanisms to E. coli, and 2> to investigate whether toxicity resulted from a real nanoparticle effect or from the release of Zn2+ in solution. To do so, both strains were submitted to various concentrations (i.e., 0.1-1mM) of nano-ZnO or Zn2+ in Luria Bertani (LB) medium. These toxicity studies take into account the nano-ZnO solubility in the culture medium by specifically monitoring the Zn2+ release in our experimental systems. In our experimental conditions, differences in tolerance to nano-ZnO or Zn2+ between both strains were clearly evidenced. W3110 is generally more tolerant to metal than MG1655, the latter showing no real difference in its sensitivity to the two zinc added forms unlike W3110. The differences in behavior between both strains could be attributed to differences in the two genomes as a mutation named amber in W3110. Moreover, by using these two closely E. coli strains, a real nano effect is here clearly demonstrated providing a model to study the toxicity of ZnO nanoparticles