A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2-carboxy-D-arabinitol 1-phosphate (CA1P), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco-bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark-adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco.z 1999 Federation of European Biochemical Societies.