Acyl carrier protein (ACP) is an essential cofactor for plant fatty acid synthesis. Three isoforms occur in barley seedling leaves. The genes Acl1 and Acl3 coding for the predominant ACP I and the minor ACP III, respectively, have been cloned and characterized as has a full-length cDNA for ACP 111. Both genes, extending over more than 2.5 kb, have a conserved mosaic structure of four exons and three introns which result in mRNAs of ca. 900 bases. Alignment of the DNA sequences demonstrates that homology is restricted to the two exons coding for the mature protein whereas the remaining segments of the genes including the transit peptide-coding domains lack homology. Southern blot analyses demonstrate that Acl1 and Acl3 represent single copy genes located on chromosomes 7 and 1, respectively. Primer extension analyses identified multiple transcription start sites in both genes. The promoter regions are remarkably different; that of Acl3 resembles those for mammalian housekeeping genes in having a high G + C content plus three copies of an RNA polymerase II recognition GC element and in lacking correctly positioned TATA boxes. These features are in accordance with the hypothesis that Acl1 is specifically expressed in leaf tissue whereas Acl3 is a constitutively expressed gene.