Increasingly, photosynthetic electron transport rate is being calculated from chlorophyll fluorescence measurements. The fluorescence signal is a complex mixture of contributions from different depths within the mesophyll. One condition required for electron transport calculated from fluorescence to represent the rate accurately is that the ratio of photosynthetic capacity to light absorbed be constant throughout the leaf. In order to explore the fluorescence properties of leaves where this assumption is not true, a new approximation for phiPSII is used to generate F'm and Fs values throughout the leaf. Fs is assumed to be proportional to the amount of light absorbed from the fluorescence measuring beam and constant, i.e. independent of the actinic irradiance or CO2 concentration. This assumption is validated by measurements from Eucalyptus maculata, Flaveria bidentis and Triticum aestivum, with two different types of fluorometer, where irradiance or CO2 response curves were measured with normal or inverted leaf orientations. The new approach enables fluorescence values to be generated at each layer in a multilayer model. Two applications using this approach are presented. First, the model is used to show that when quantum yield varies through a leaf, then fluorescence will lead to an incorrect estimate of electron transport rate. Secondly, since chlorophyll fluorescence is also used to calculate the CO2 concentration at the sites of carboxylation within chloroplasts, Cc, the model is also used to show that Cc may vary with depth. Significant variation in Cc through the mesophyll could lead to an apparent dependence of internal conductance on irradiance or CO2.