Abstract: In a previous report it was shown that the activity of coupling factors isolated from chloroplasts of other plant species was higher than those obtained from chloroplasts of the same species for restoring the cyclic photophosphorylation activity of the deficient particles. However, the coupling factors then used were crude extracts or extracts after partial purification only. Verification of the results with purified preparation was highly desirable. To this end several methods of extraction and purification of chloroplast coupling factors were compared. It was found that the coupling factors extracted with dilute EDTA solution from the chloroplasts after the removal of heterogeuous proteins showed a single band on polyacrylamide gel electrophoresis. When deficient particles from spinach chloroplasts were incubated with coupling factors purified in this way from broad bean (Vicia faba L.) or barley (Hordeum vulgare L.), the restored photo- phosphorylation activity was still higher than that incubated with coupling factors prepared from spinach chloroplasts and purified in the same way. Thus it can be concluded that the enhancement effect of coupling factors from different species was due to the coupling factors per se and not to any substance(s) contaminated in the crude extracts.