The development of a specific and sensitive radioimmunoassay for the detection of zolpidem and its metabolites in urine and serum samples is described. The assay can be used to pre-screen forensic and emergency samples. 6-Methyl-2-(4-methylphenyl)imidazo[1,2-a]-pyridine-3-acetic acid was prepared as a hapten and was coupled directly and indirectly to bovine serum albumin. The immunization of rabbits with the hapten-bovine serum albumin conjugates resulted in the production of highly specific antibodies, showing no significant cross-reactivities towards existing drugs. The four-parameter logistic model was used to process the calibration data into a fitting curve (r2 = 0.9764). Intra- and inter-assay relative standard deviations were < 6.30 and < 12.28%, respectively. The limit of quantification was 0.1 ng ml-1. Using this assay, zolpidem levels were determined in urine and serum, and could be easily detected up to 48 h after oral intake of 10 mg of zolpidem.