[NiFe]-hydrogenases catalyze the oxidation of H2 to protons and electrons. This reversible reaction is based on a complex interplay of metal cofactors including the Ni–Fe active site and several [Fe–S] clusters. H2 catalysis of most [NiFe]-hydrogenases is sensitive to dioxygen. However, some bacteria contain hydrogenases that activate H2 even in the presence of O2. There is now compelling evidence that O2 affects hydrogenase on three levels: 1) H2 catalysis, 2) hydrogenase maturation, and 3) H2-mediated signal transduction. Herein, we summarize the genetic, biochemical, electrochemical, and spectroscopic properties related to the O2 tolerance of hydrogenases resident in the facultative chemolithoautotroph Ralstonia eutropha H16. A focus is given to the membrane-bound [NiFe]-hydogenase, which currently represents the best-characterized member of O2-tolerant hydrogenases.