The purpose of these studies was to evaluate the performance characteristics of the Cozart Microplate Enzyme Immunoassay (EIA) for the determination of methadone in oral fluid from patients in a drug misuse treatment program. Oral fluid specimens were collected using the Cozart RapiScan Collection system from 198 donors who were receiving treatment for their addiction and were monitored for drug misuse. Oral fluid specimens were also collected from forty volunteer donors who were not drug users. The specimens were analyzed in the laboratory by EIA and then analysed for methadone and its main metabolite EDDP by gas chromatography-mass spectrometry (GC-MS). A total of 103 samples were confirmed positive for methadone. The Cozart Microplate EIA for d-Methadone in oral fluid using a cutoff of 30 ng/mL in diluted oral fluid had a sensitivity of 91.3% +/- 2.8% and a specificity of 100% +/- 1.0% vs. GC-MS.