Measurement of the chlorophyll (Chl) a fluorescence rise (FR) under higher exciting irradiance (EI), the O-J-I-P transient, or under lower irradiance, the O-I-P transient, is a routinely used method to access photosystem 2 function in thylakoid membranes of chloroplasts. Our measurements with a suspension of pea thylakoid membranes showed that the relative heights of the J and I steps in the FR depended not only on EI but also on the concentration and thickness of the sample. We explain this effect as a consequence of the gradient of EI within the sample. We tested this suggestion by theoretical simulations of the FR based on the model that was previously used for simulation of the FR considering in addition the gradient of EI within the sample. Our theoretical results correspond well with the experiments. The irradiance gradient effect may influence measured FR significantly and this fact should be taken into consideration in the interpretation of measured FRs. and P. Sušila ... [et al.].