Cotton ovules collected during late September with prevalent night cool temperature (15 "C), cultured at 30 "C/15 "C i.e. cycling temperatures in Beasley and Ting medium hadvery few epidermal cells showing bulging. Supplementing cultures with guaianolide derivative I (E-13 methyldehydrocostus lactone) promoted fibre initiation. At -1 day preanthesis (DPA), IAA oxidase activity declined in guaianolide-treated cultures but increased during the elongation phase and was enhanced during the secondary wall thickening phase. However, o-diphenol oxidase activity was adversely affected during the fibre initiation phase. The activities of all the other enzymes studied viz. acid invertase, phenylalanine ammonia lyase, /3-glucosidase and IAA oxidase increased, except ,@-galactosidase, during the later phase in comparison with the controls. The present study indicates that guaianolide derivative I triggers early initiation and promotes fibre elongation by regulating o-diphenols and IAA-oxidation levels, which in turn check wall loosening. Considerable enhancement in the soluble acid invertase activity by this compound suggests its role in apoplastic sucrose hydrolysis, thereby preventing its accumulation.Abbreviations: DPA = days pre anthesis; DAC = days after culturing; DAA = days after anthesis.