Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When beta-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates palmitoyl-ACP to beta-ketostearoyl-ACP but has no activity towards decanoyl-ACP. The first protein is designated beta-ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second protein is designated beta-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification of synthase II is described, and its activity is compared to synthase I. Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis.