Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of a modified strain of Synechocystis sp. PCC 6803 that contains a thioesterase and from which the acyl-ACP synthetase has been deleted. Using ammonium sulfate precipitation to isolate a crude protein fraction containing ACP, immunoblot analysis was performed to determine relative amounts of free and acylated-ACP in the cell. The nature of fatty acids attached to ACP was determined by creating butylamide derivatives that were analyzed using GC/MS. Immunoblot analysis showed a roughly 1:1 ratio of acylated ACP to free ACP in the cell depending on the nutritional state of the cell. From GC/MS data it was determined that palmitic acid was the predominate component of acyl groups attached to ACP. The results indicate that there is a significant amount of acyl-ACP, a feedback inhibitor of early steps in the fatty acid biosynthesis pathway, in the cell. Moreover, the availability of free ACP may also limit fatty acid biosynthesis. Most likely it is necessary for ACP to be overexpressed or to have the palmitic acid cleaved off in order to synthesize optimal amounts of lauric acid to be used for cyanobacterial biofuel production.