The cross-reactivity of an immunological probe to the key photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) was characterized as part of a larger effort to determine maximal photosynthetic rates of individual phytoplankton cells. Polyclonal antiserum was produced against purified Rubisco from the marine diatom Chaetoceros gracilis. The results of western immunoblotting demonstrated that the antiserum reacted positively with Rubisco from 38 species of algae and higher plants and failed to react with only three species of dinoflagellates and one prochlorophyte species. However, the binding affinity or the strength of the cross-reaction for the polyclonal antiserum with purified Rubisco varied among species. The antiserum was then affinity purified against spinach Rubisco and its binding affinity for purified Rubisco determined by ELISA. Two taxonomic groupings resulted: one with high-binding affinity (these species included chrysophytes, bacillariophytes, prymnesiophytes, and chlorophytes) and the other with low-binding affinity (dinophytes and cyanophytes). Rubisco concentration per cell and light-saturated rates of photosynthesis were highly correlated for cultures of the diatom Tha/ussiosira weissfogii. These results indicate that affinity-purified antiserum can be rigorously characterized for use in quantifying Rubisco concentration and for assessing the maximal photosynthetic potential of individual phytoplankton cells.