The cholesterol-depleting drug methyl-β-cyclodextrin (Me-β- CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5–50 mM Me-β-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-β-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [³H]cholesterol, 30–90 min of treatment with 5–50 mM Me-β-CD removed 50%–70% of cell- associated label, and cholesterol depletion was not observed with α-cyclodextrin. After long-term (20–23 h) labeling of oocytes with [³H]cholesterol, Me-β-CD treatment resulted in dose- dependent cholesterol depletion in the 5–50 mM range, and 50 mM Me-β-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5–50 mM Me-β-CD also decreased recovery of low-density membrane by detergent- free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.