Estimates of thylakoid electron transport rates (J(e)) from chlorophyll fluorometry are often used in combination with leaf gas exchange measurements to provide detailed information about photosynthetic activity of leaves in situ. Estimating J(e) requires accurate determination of the quantum efficiency of Photosystem II (Phi(P)), which in turn requires momentary light saturation of the Photosystem II light harvesting complex to induce the maximum fluorescence signal (F(M)'). In practice, full saturation is often difficult to achieve, especially when incident photosynthetic photon flux density (Q) is high and energy is effectively dissipated by non-photochemical quenching. In the present work, a method for estimating the true F(M)' under high Q was developed, using multiple light pulses of varying intensity (Q'). The form of the expected relationship between the apparent F(M)' and Q' was derived from theoretical considerations. This allowed the true F(M)' at infinite Q' to be estimated from linear regression. Using a commercially available leaf gas exchange/ chlorophyll fluorescence measurement system, J(e) was compared to gross photosynthetic CO(2) assimilation (A(G)) under conditions where the relationship between J(e) and A(G) was expected to be linear. Both in C(4) leaves (Zea mays) in ambient air and also in C(3) leaves (Gossypium hirsutum) under non-photorespiratory conditions the apparent ratio between J(e) and A(G) declined at high Q when Phi(P) was calculated from F(M)' measured simply using the highest available saturating pulse intensity. When F(M)' was determined using the multiple pulse / linear regression technique, the expected relationship between J(e) and A(G) at high Q was restored, indicating that the Phi(P) estimate was improved. This method of determining F(M)' should prove useful for verifying when saturating pulse intensities are sufficient, and for accurately determining Phi(P) when they are not.