A novel HPLC method for simultaneous determination of estradiol and its seven degradation products in topical gel was developed. Zorbax SB-CN (150 mm x 4.6 mm, 5 microm) analytical column and mobile phase composed of acetonitrile, phosphoric acid 0.085%, and tetrahydrofurane (27:63:10, v/v/v) at flow-rate 1.0 ml min(-1) were used for the chromatographic separation using UV detection at 225 nm. The active substance estradiol was separated from all its known degradation products successfully. Two degradation products estrone and Delta(9(11))-estrone were not separated sufficiently, their peaks were evaluated as a sum of two components. The method was validated according to ICH guideline recommendations and thereafter it was successfully applied for stability tests of topical cream Estrogel HBF in the quality control laboratory. Limits of detection for degradation products ranged from 1.03 x 10(-5) to 1.14 x 10(-4) mg ml(-1), limits of quantitation for degradation products were in the range 3.43 x 10(-5) to 3.81 x 10(-4) mg ml(-1). The developed method is selective, precise, accurate and sensitive enough for determination of estradiol and its known degradation products.