This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids.