Background: It has been suggested that oxygen (O-2) diffusion could be favored in water enriched in O-2 by a new electrolytic process because of O-2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O-2 and facilitate O-2 diffusion along PO2 gradients. Materials and methods: Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O-2 by injection or the electrolytic process 1) at an O-2 concentration decreasing from 240 mu mol/L to 10 mu mol/L (132 mmHg to 5 mmHg), with glutamate-malate or N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate-malate as substrate. Results: As expected, maximal respiration decreased with O-2 concentration and, when compared to glutamate-malate, the apparent Km O-2 of mitochondria for O-2 was significantly lower with TMPD-ascorbate with both waters. However, when compared to the water enriched in O-2 by injection, the Km O-2 was significantly lower with both electron donors in water enriched in O-2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. Conclusion: In this experiment, a higher affinity of the mitochondria for O-2 was observed in water enriched in O-2 by electrolysis than by injection. This observation is consistent with the hypothesis that O-2 diffusion can be facilitated in water enriched in O-2 by the electrolytic process.