A case of a clinically significant interaction between a fluoroquinolone antimicrobial agent and metal cations was first reported in 1985. The hypothesized mechanism - decreased fluoroquinolone intestinal permeability due to complex formation between metal cations and ciprofloxacin - was based ona 1978 work with nalidixic acid. While clinical research and numerous in vitro physico-chemical and chelation chemistry studies of fluoroquinolone-metal cation combinations simply accepted this explanation, the few in vitro studies, which were aimed to investigate the nature of the interaction mechanism, provided conflicting results. This was most likely due to the sensitivity of the interaction to in vivo conditions, which were not reproduced in vitro. All the above-mentioned studies including our earlier work in vitro were performed with diluted solutions of fluoroquinolones and metal cations. Now we provide results obtained on rat intestine in side-by-side diffusion chambers with saturated solutions of fluoroquinolones and metal cations in the donor compartment as it is most likely in the human small intestine in vivo. The fluoroquinolone permeability decreased under these conditions in the presence of metal cations and the obtained results show that the ciprofloxacin-aluminum complex does not permeate the intestinal mucosal membrane.