In the present study, a novel, fast, sensitive and robust method to quantify ethinylestradiol in human plasma using 17alpha-ethinylestradiol-d4 as the internal standard (IS) is described. The analyte and the IS were extracted from acidified plasma by liquid-liquid extraction (LLE) using diethyl ether-hexane followed by online solid phase extraction (SPE) using online C18 cartridges. Extracted samples were analyzed by high-performance liquid chromatography coupled to atmospheric pressure photoionization tandem mass spectrometry (HPLC-APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 microm analytical column. The method had a chromatographic run time of 2.50 min and a linear calibration curve over the range 5-500 pg ml(-1) (r(2)>0.9992). The lowest concentration quantified was 5 pg ml(-1), demonstrating acceptable accuracy and precision. The intra-assay precisions ranged from 2.1 to 14.6%, while inter-assay precisions ranged from 4.4 to 11.4%. The intra-assay accuracies ranged from 94.6 to 103.8%, while the inter-assay accuracies ranged from 98.9 to 101.6%. The recovery of ethinylestradiol was determined as part of the assay validation process and was 73.1 and 79.0% for the concentrations 15 and 375 pg ml(-1), respectively. Short-term stability showed that ethinylestradiol was stable in plasma for at least 19 h at room temperature or for at least 385 days when stored at -20 degrees C. In the study of bioequivalence conducted in Brazil, healthy volunteers received two ethinylestradiol 0.035 mg tablet formulations using an open, randomized, two-period crossover design with a 2-week washout interval. Since the 90% confidence interval for C(max) and area under the curve ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the two ethinylestradiol formulations are bioequivalent with respect to both the rate and the extent of absorption.