Phospholipase Dalpha (PLDalpha) was isolated from cultivated cotton (Gossypium hirsutum) and characterized. Two PLDalpha genes were identified in the allotetraploid genome of G. hirsutum, derived from its diploid progenitors, G. raimondii and G. arboreum. The genes contained three exons and two introns. The translated products shared a 98.6% homology and were designated as GrPLDalpha and GaPLDalpha. Their ORFs encoded a polypeptide of 807 amino acids with a predicted molecular mass of 91.6 kDa sharing an 81-82% homology with PLDalpha1 and PLDalpha2 from A. thaliana. A possible alternative splicing event was detected at the 5' untranslated region which, however, did not result in alternative ORFs. Cold stress (10 degrees C or less) resulted in gene induction which was suppressed below control levels (25 degrees C or 22 degrees C growth temperature) when plants were acclimated at 17 degrees C before applying the cold treatment. Differences in the expression levels of the isoforms were recorded under cold acclimation, and cold stress temperatures. Expression was light regulated under growth, acclimation, and cold stress temperatures. Characterization of the products of lipid hydrolysis by the endogenous PLDalpha indicated alterations in lipid species and a variation in levels of the signalling molecule phosphatidic acid (PA) following acclimation or cold stress.