In 16 medical examiner's cases, which were found to be barbiturate-positive by thin-layer chromatographic screening of the liver, blood barbiturate concentrations were determined by gas chromatography. The corresponding vitreous humor samples were screened by the enzyme multiplied immunoassay technique, the EMIT-st serum barbiturate assay. By using the recommended dilution for detecting serum barbiturates, it was possible to detect barbiturates in vitreous humor at a toxic concentration. By using one fourth the amount of diluent, the barbiturates could be detected also at a therapeutic concentration. The EMIT-st assay proved to be useful in the screening for barbiturates in vitreous humor, a material that is readily available in forensic toxicology.