An enzyme-linked immunosorbent assay (ELISA) for quantitation of urinary fentanyl was evaluated as a screening tool for detecting abuse of this potent narcotic. The assay was found to have reproducible calibration curves from 0.5 to 10 ng/mL and a limit of detection of 0.5 ng/mL. Interference by proteins, glucose, or pH was negligible. The assay was specific for fentanyl with little cross-reactivity against despropionyl fentanyl and norfentanyl metabolites, other analgesics and common drugs of abuse. To evaluate its use in humans, urines were collected from 57 normal individuals, 48 patients seen in the Emergency Department, and 18 surgical patients receiving either low (50 micrograms) or moderate fentanyl dosage (200 and 250 micrograms) for routine anesthesia. In patients receiving 50 micrograms (a dose consistent with early abuse), urinary fentanyl was detectable for 3 to 10 h post administration. In patients receiving 200 or 250 micrograms (a dose more consistent with addiction), urinary fentanyl was detectable for longer time periods (> 24 h). These results indicate that the ELISA is sensitive for the detection of recent fentanyl exposure under conditions likely to mimic those in abuse and addiction. The assay is simple to perform, reliable, and can be used to screen urine specimens prior to gas chromatography/mass spectrometry (GC/MS) confirmation.