Remifentanil is an ultra-short-acting opioid under evaluation for use during surgical procedures which require opioid analgesia or anesthesia. It has an N-substituted methyl propanoate ester group which is highly susceptible to clevage by blood and tissue esterases as well as to chemical hydrolysis. A selective and specific high performance liquid chromatography method was developed to quantitate remifentanil in rat blood. A liquid-liquid extraction method using n-butyl chloride was used to separate interfering endogenous products from the compound of interest. Reverse phase chromatography with ultraviolet (lambda 210 nm) detection was used to quantitate the eluate. The calibration curves were found to be linear in the range 2.5-250 ng ml-1. Intra-day assay variability was 15% or less for all standards evaluated. The method was applied to blood samples collected from a short-term infusion study in rats. Good recovery, linearity, accuracy and precision were achieved with the assay.