Acetyl coenzyme A (CoA) biosynthesis in spinach chloroplasts has been investigated by following the incorporation of bicarbonate and acetate into fatty acids under a variety of conditions. Both substrates were readily incorporated into fatty acids in a light-dependent manner by intact photosynthesising chloroplasts, but when the concentrations of these substrates were adjusted to those found in vivo, i.e. 200 μM acetate, 10 μM bicarbonate, then acetate was found to supply carbon atoms for fatty acids biosynthesis via acetyl CoA at forty times the rate of bicarbonate. It is proposed that extra-chloroplastic free acetate is the pricipal substrate for chloroplasts acetyl CoA biosynthesis in spinach.