Ethylene is a major stimulating factor for natural rubber production in Hevea brasiliensis and is often applied in the form of Ethephon, an ethylene releaser. In a positive feedback type mechanism, the application of ethylene leads to the enhanced expression of genes involved in ethylene biosynthesis such as ACC oxidases, which are also responsive to wounding. We studied the regulation of ethylene biosynthesis in rubber trees through the characterisation of the ACC oxidase multigene family and the responses of individual genes to ethylene and wounding. Three full-length cDNAs, HbACO1, HbACO2 and HbACO3, were isolated. The HbACOs were 1115, 1174 and 1074 bp long, respectively, with open reading frames encoding polypeptides of 312, 318 and 318 amino acids, respectively. The genomic sequences of HbACO1 and HbACO2 were also characterised, revealing divergent gene structures: HbACO1 (1456 bp) possesses three introns and four exons, while HbACO2 (1418 bp) consists of two introns and three exons. All of the HbACO genes were expressed at all stages of development studied, from in vitro callus to the exploited plant, but with different expression profiles. Nevertheless, no expression was detected in latex. In the bark of the shoot of the first flush of budded plants, HbACO1 was expressed at a higher level than the other genes, and was down-regulated by ethylene and wounding in both leaf and bark tissues. In contrast, HbACO2 and HbACO3 were transiently induced in response to ethylene and wounding. Treatment with 1-MCP, an ethylene action inhibitor, abolished the ethylene induction of HbACO2 gene, showing that this gene was under positive feedback regulation. The expression of HbACO3 gene was also induced by ethylene treatment and wounding, again with an antagonistic effect of 1-MCP. In conclusion, HbACO1 appears to be responsible for basal levels of ethylene production while HbACO2 and HbACO3 are up-regulated in response to external factors.