We introduce a new, non-invasive technique to measure linear electron transfer in intact leaves under steady-state illumination. Dark-interval relaxation kinetic or 'DIRK' analysis is based on measurements of the initial rates of relaxation of steady-state absorbance signals upon a rapid light-dark transition. We show that estimates of electron flux by DIRK analysis of absorbance signals, reflecting redox changes in the photosynthetic electron transfer chain, can yield quantitative information about photosynthetic flux when the light-dependent partitioning of electrons among redox components of the electron transfer chain are considered. This concept is modeled in computer simulations and then demonstrated in vivo with tobacco plants under non-photorespiratory conditions resulting in linear relationships between DIRK analysis and gross carbon assimilation (A(G)). Estimation based on DIRK analysis of the number of electrons transferred through the photosynthetic apparatus for each CO(2) fixed was within 20% of the theoretical value. Possible errors and future improvements are discussed. We conclude that the DIRK method represents a useful tool to address issues such as plant stress and photosynthetic regulation.