The dynamics of sulphide oxidation in an experimental biofilm of the green sulphur bacterium, Prosthecochloris aestuarii, were studied using a newly developed light–dark cycling procedure. The biofilm was grown for 6 weeks in a benthic gradient chamber, in which gradients of light, sulphide and oxygen were imposed experimentally. The H₂S concentrations and pH were measured with microsensors as a function of depth in the biofilm and of time after a change in illumination status. The sulphide oxidation rates were calculated as a function of time and depth in the biofilm using a numerical procedure to solve the non-stationary general diffusion equation. A close agreement was found between the areal rates of anoxygenic photosynthesis during the cycling procedure and the steady state before the cycling experiment. For the different layers of the biofilm, the maximum activity was observed after 10–12 min of light exposure. After this maximum, sulphide oxidation decreased concomitantly with sulphide concentration, indicating sulphide limitation of anoxygenic photosynthesis. This lag time limits the application of the standard dark–light shift method with a brief light exposure of a few seconds and, therefore, the numerical procedure described in this study enables the depth distribution of anoxygenic photosynthesis rates in microbial mats to be determined more accurately.