A sensitive and specific gas chromatographic-mass spectrometry with the extracted ion chromatograms (GC–MS/EIC) method has been developed and validated for the identification and quantification of penehyclidine (PH) in human and animal blood. The chromatography was on HP-5 capillary column (12m×0.2mm×0.3µm). PH-d5 was selected as the internal standard (IS). Simultaneous MS detection of PH and IS was performed at m/z 315 (PH) and m/z 320 (PH-d5), and the EIC of the two compounds was at 175 and 180. PH and PH-d5 eluted at approximately 8.2 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 0.1–100 ng/ml in the blood. The lower limit of quantification (LLOQ)was reproducible at 50 pg/ml in both human and animal blood. The within-day and between-day precisions were no more than 9.1%. This method was successfully applied to quantification and pharmacokinetic studies of PH in the subjects. The concentration–time profiles of PH in humans, rabbits and mice were all fitted to first order absorption two-compartment open model after i.m. a single dose. The differences in absorption, distribution and elimination of PH among the species were found. The results provided the important information for developing a novel anti-cholinergic drug and for obtaining a more effectual remedy in clinical practice.