Cotton fibres are hair-like single-cells that elongate to several centimetres long after their initiation from the ovule epidermis at anthesis. The accumulation of malate, along with K+ and sugars, is thought to play an important role in fibre elongation through osmotic regulation and charge balance. However, there is a lack of evidence for or against such an hypothesis. Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme responsible for the synthesis of malate. The potential role of PEPC in cotton fibre elongation is examined here. Developmentally, PEPC activity was higher at the rapid elongation phase than that at the slow elongation stage. Genotypically, PEPC activity correlated positively with the rate of fibre elongation and the final fibre length attained. Importantly, suppression of PEPC activity by LiCl that reduces its phosphorylation status decreased fibre length. To examine the molecular basis underlying PEPC activity, two cDNAs encoding PEPC, GhPEPC1 and 2, were cloned, which represents the major PEPC genes expressed in cotton fibre. RT-PCR analyses revealed that GhPEPC1 and 2 were highly expressed at the rapid elongation phase but weakly at the slow-to-terminal elongation period. In situ hybridization detected mRNA of GhPEPC1 and 2 in 1 d young fibres but not in the ovule epidermis prior to fibre initiation. Collectively, the data indicate that cotton fibre elongation requires high activity of PEPC, probably through the expression of the GhPEPC1 and 2 genes.