Culturing mammalian cells in a hypoxic environment is a useful model to study mechanisms of hypoxia associated diseases. Low solubility of O2 in aqueous solutions and slow diffusion from the gas phase through the culture medium to cells is the major barrier to rapid changes in O2 concentration. The oxygen concentration to which cells are actually exposed results from the balance between O2 diffusion through culture medium and rate of O2 consumption by the cells. It may take several hours to fully equilibrate culture media with a gas of defined composition when regular culture dishes are used. Time depends on thickness of the layer of medium and surface area. Mixing or use of gas-permeable flasks shortens equilibration. For hypoxic exposure cells can be kept in a plastic box flooded with gas of the required composition, or in oxygen- and CO2 controlled incubators or glove boxes. If fast responses to hypoxia are studied, culture medium should be replaced with one equilibrated to gas of the required composition. An important aspect is handling of cells that were exposed to hypoxia for harvesting or further experimentation. The procedure depends on the readout. If changes in parameters to be tested are readily reversible upon reoxygenation (e.g. ROS, nuclear HIF) then cells have to be handled in an hypoxic environment such as in an environmental glove box or under a stream of gas. If changes are stable (e.g. cell protein content and slow turnover) cells might even be handled in normoxia.