A reagent strip has been developed for measuring phenobarbital concentrations in serum or plasma. This strip utilizes the apoenzyme reactivation immunoassay system (ARIS) and is designed for use with the Ames Seralyzer reflectance photometer. The test takes 85 s and compares strip reactivities with a two-point calibration line that is stored in the instrument. Within-run precision coefficient of variation ranges from 2.4 to 4.1% over five concentrations while the between-run precision ranges from 2.3 to 4.9%. Results obtained with clinical serum samples correlated well (r greater than 0.99) with those obtained by a fluorescence polarization immunoassay (TDx). The strips crossreact with other barbituric acid derivatives that are not coadministered with phenobarbital. Crossreactivity to the metabolite p-hydroxyphenobarbital is sufficiently low as to not interfere in the usual specimens. Samples that are hemolyzed, highly icteric, or from uremic patients should be avoided. This assay is a rapid and convenient method for monitoring serum or plasma phenobarbital concentrations. It is particularly well suited for decentralized test sites, such as emergency rooms, urgent care centers, and physician's offices.