The accumulation of methylphenobarbital (MPB) and phenobarbital (PB) in plasma in two volunteers who were given continuous once-daily oral doses of MPB for 3 weeks was demonstrated by use of a selected ion-monitoring GC/MS assay. It was shown that the PB concentration exceeded the MPB concentration in plasma after about day 4, and that both barbiturates achieved plateau concentrations after about 2 weeks. GC/MS studies on the urine of these volunteers permitted the identification of several new metabolites of MPB. These included 5-ethyl-5-(4-hydroxyphenyl)-1-methylbarbituric acid (p-OH-MPB), and the 3-O-methylcatechols of both MPB and PB. The meta-isomers of hydroxy-MPB and hydroxy-PB were identified in urine extracts, but were shown to be methodological artifacts. Quantitative studies, with use of a HPLC assay, were carried out for p-OH-MPB, PB and p-OH-PB in urine, and it was shown that these three substances collectively accounted for some 50% of the administered dose (greater than 30% as p-OH-MPB).