Our current understanding of the role of phytohormones in the development of cotton fibers is derived largely from an amenable culture system in which cotton ovules, collected on the day of anthesis, are floated on liquid media. Under these conditions, supplemental auxin and gibberellin were found to promote fiber initiation and elongation. More recently, addition of low concentrations of the brassinosteroid brassinolide (BL) were also found to promote fiber elongation while a brassinosteroid biosynthesis inhibitor brassinazole2001 (Brz) inhibited fiber development. In order to elucidate the role of brassinosteroid in cotton fiber development further, we have performed a more detailed analysis of the effects of these chemicals on cultured cotton ovules. Our results confirm that exogenous BL promotes fiber elongation while treatment with Brz inhibits it. Furthermore, treatment of cotton floral buds with Brz results in the complete absence of fiber differentiation, indicating that BR is required for fiber initiation as well as elongation. Expression of fiber genes associated with cell elongation increased in ovules treated with BL and was suppressed by Brz treatment, establishing a correlation between brassinosteroid-regulated gene expression and fiber elongation. These results establish a clear connection between brassinosteroid and fiber development and open the door for genetic analysis of cotton development through direct modification of the brassinosteroid signal transduction pathway.