Chlorophyll a accumulated when chlorophyllide b was incubated with isolated cucumber etioplasts in the dark. When [C-14]chlorophyllide b was used as the substrate for chlorophyll synthesis, [C-14]chlorophyll a was formed, showing that chlorophyll a was synthesized from the exogenously added chlorophyllide b, not by light-independent reduction of endogenous protochlorophyllide. The reaction studied showed an optimal pH of 7.5 and required both the soluble and membrane fractions of etioplasts together with ATP. Time course experiments showed that chlorophyll a began to accumulate later than chlorophyll b. Chlorophyllide b esterified with geranylgeraniol accumulated first, but there was little accumulation of chlorophyll a with an unhydrogenated prenyl side chain. No chlorophyllide a was detected during incubation. These observations indicate that esterified chlorophyll b was converted to chlorophyll a. This conversion would play an important role in the use of chlorophyll b for the synthesis of chlorophyll a in the reconstruction of photosystems.