A procedure is described which permits determination of the absolute absorption cross-section of a photosynthetic unit from the kinetics of reaction center photo-oxidation under weak, continuous actinic illumination. The method was first tested on a simple model compound of known absorption cross-section. We then applied the technique to absorption cross-section and functional antenna size measurements in photosystem I (PS I). A kinetic model is presented that can be used to fit P700 photo-oxidation measurements and extract the effective photochemical rate constant. The procedure is shown to properly correct for sample scattering and for the presence of heterogeneous absorbers (pigments not functionally coupled to P700). The relevance of these corrections to comparisons of antenna size using techniques that measure 'relative' absorption cross-sections is discussed. Measurements on pea thylakoids in the presence and absence of 5 mM MgCl2 show a 45% increase in PS I absorption cross-section in unstacked thylakoids. Analysis of detergent-isolated 'native' PS I preparations (200 chlorophyll a+b/P700) clearly indicate that the preparation contains a broad distribution of antenna sizes. Finally, we confirm that Chlamydomonas reinhardtii strain LM3-A4d contains a PS I core antenna complex which binds only ∼60 chlorophyll a/P700, about half the functional size of the wild type complex. Limitations associated with calculation of functional antenna size from cross-section measurements are also discussed.