Transcripts from immature fibers and stripped ovules (fibers removed) of cotton (Gossypium hirsutum L.) were compared by differential display to identify cDNA fragments that represent mRNAs that are expressed primarily in cotton fibers. Eight independent fiber-specific cDNA fragments were isolated. One of these cDNAs had strong sequence similarity with acyl carrier protein (ACP). A full-length cDNA for the cotton fiber-specific ACP was isolated using a PCR cDNA library screening technique. This 713 bp cDNA has an open reading frame that encodes a 136 amino acid polypeptide. Overall nucleotide and amino acid sequence identities with other plant ACP gene sequences averaged 66% and 60% respectively. A 19 amino acid sequence surrounding the prosthetic group attachment site is nearly identical to other plant ACP genes. Northern blot analyses showed that transcripts homologous to this fiber-specific ACP cDNA were predominantly expressed during the elongation stage of fiber development. Initial genomic Southern blot analysis indicated that a single copy of the fiber-specific ACP gene may be present in both the cotton A and D genomes, since diploid Gossypium species with A or D genomes gave identical bands. We speculate that this putative fiber-specific ACP may play an important role in rapidly elongating cotton fibers by contributing to the synthesis of membrane lipids. It is also apparent that during the evolution of cotton a member of the ACP gene family has been recruited for specific expression in cotton fibers.