An analytical method was developed and validated for the quantitative determination of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), a novel heat shock 90 inhibitor, in human plasma. Calibration curves were linear in the concentration range of 1-500 ng/mL. Sample pretreatment involved a liquid-liquid extraction of 0.2 mL aliquots of plasma with ethyl acetate. 17-DMAG and the internal standard, beclomethasone, were separated on a Zorbax SB C18 column (75 mm x 2.1 mm, 3.5 microm), using a mobile phase composed of methanol and 0.2% formic acid (55:45, v/v). The column effluent was monitored by mass spectrometry with electrospray ionization. For the quality control samples at four different concentrations that were analyzed in quintuplicate, on four separate occasions, the accuracy and precision ranged from 93.8% to 99.5% and 1.4% to 3.3%, respectively. The assay modifications significantly improve upon our original, validated method. The developed method was subsequently applied to study the pharmacokinetics of 17-DMAG in a group of 23 patients.