The activation state of the chloroplast stromal enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-5-phosphate kinase was assayed by injecting the chloroplasts into an assay medium where the chloroplasts are immediately lysed and the enzymic assay is terminated 15–30 s later. (1) After the onset of illumination the activities of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase rise 20- and 30-fold, respectively, and rapidly decrease again when the light is turned off. The light activation of both enzymes shows a substrate requirement. The catalytic activity of the activated enzyme is dependent on the pH and the Mg 2+ concentration in the assay medium, with the fructose-1,6-bisphosphatase more markedly affected. (2) The light activation of ribulose-5-phosphate kinase occurs even more rapidly than that of the other two enzymes and leads to about 10-fold activation. Moreover, the inactivated form of the enzyme, as extracted from chloroplasts in the dark, is strongly inhibited when ADP is added to the assay medium. (3) These data clearly indicate that chloroplasts have the ability to inactivate the three mentioned Calvin cycle enzymes virtually completely in the dark phase by a combined effect of changes of the reductive state of the electron-transport carrier on all three enzymes, changes of stromal Mg 2+ and pH on fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase and changes of stromal ATP ADP ratios on ribulose-5-phosphate kinase.