Cotton fiber developing on cultured ovules is an important experimental tool, even though in vitro grown fibers are shorter than in situ grown fibers. Fiber measurements and microscopy show that early fiber elongation increased when calcium concentration in the culture medium was reduced from the traditional 3 mM to 0.1 mM. Fiber length in 0.1 mM calcium responded negatively to trifuoperazine, which antagonizes calcium-dependent calmodulin signaling. Fiber length in 0.1 mM calcium responded positively to calcimycin, which antagonizes intracellular calcium compartmentalization. In contrast, neither compound affected the length of fibers in 1 mM calcium medium. Transferring ovules between low- and high-calcium concentration media showed that fiber elongation was most sensitive to exogenous calcium concentration in the first 1 to 2 d after anthesis (2-3 d in culture). The transcript level of a fiber expansin gene increased when ovules were cultured in low-calcium medium. These data show that use of low-calcium medium in the culture of ovules less than 3 d after anthesis promoted fiber elongation through effects related to intracellular calcium signaling. Future in vitro experiments probing the controls of fiber development can be conducted with less restriction on elongation imposed by the culture medium, which should help to uncover more of the mechanisms controlling fiber development.