In vitro photodynamic therapy experiments are usually performed by irradiating cells in confluent or nearly confluent monolayer cultures. Oxygen is consumed in the monolayer by photodynamic reactions and cellular respiration and is supplied by diffusion from the overlying medium. Calculations of oxygen concentration by numerical solution of the time-dependent diffusion equation show that hypoxia can be induced in the monolayer under typical PDT conditions and that this will limit the total treatment effect. There is an optimum fluence rate at which the greatest singlet oxygen dose can be delivered before hypoxia becomes limiting. It is recommended that researchers use these calculations to avoid hypoxia or confirm that fortuitous oxygen transport by other mechanisms (e.g., convection) is adequate to prevent it.