An Arabidopsis thaliana partial cDNA was previously identified with a sequence similar to the lauroyl-acyl carrier protein (ACP) thioesterase from Umbellularia california (Grellet et al., 1993, Plant Physiol. Biochem. 31, 599-602). Using this DNA fragment, we isolated a 1.8-kb cDNA coding for a 412-amino-acid preprotein. The deduced amino acid sequence is 51% identical to the lauroyl-ACP thioesterase but only 39% identical to safflower oleoyl-ACP thioesterase. The cDNA was expressed in Escherichia coli and the gene product showed thioesterase activity for long-chain acyl-ACPs (14:0, 16:0, 18:0, 18:1 delta 9cis). When expressed in beta-oxidation mutants of E. coli, lipid analysis revealed that cells transformed with the thioesterase produced high amounts of free fatty acids that mostly consisted of 16:0 and some 14:0, 16:1 delta 9cis, and 18:1 delta 11cis. Antibodies were raised to the recombinant protein and used to determine tissue-specific and developmental expression in A. thaliana and Brassica napus. A 40-kDa protein was detected by immunoblots in A. thaliana siliques, leaves, and roots. A maximal expression of the B. napus protein between 18 and 31 days after flowering was found, which correlates with the rapid accumulation of triacylglycerols in the seeds. Based upon these results, we suggest that this long-chain acyl-ACP thioesterase may be a ubiquitous enzyme in plants which is involved in the synthesis of long-chain fatty acids.