In plasma of untreated male Wistar rats the mean free fraction of desmethylimipramine (DMI) amounted to 10.5%. A five-day oral treatment with phenobarbital (PB) reduced it to 6.2%. A similar effect was produced in female Wistar rats and in male, but not in female Sprague-Dawley rats. Pretreatment with DDT did not alter DMI binding. The PB effect not be attributed to the presence of PB or its metabolites in plasma nor to lower levels of endogenous compounds attached to plasma proteins. Studies on single DMI-binding proteins in plasma of male Wistar rats disclosed unchanged concentrations and binding properties of albumin and total lipoproteins following PB administration, while alpha 1-acid glycoprotein isolated from plasma of PB-treated animals bound DMI stronger than that from controls and contained a higher percentage of N-acetylneuraminic acid. The enhanced binding to a chemically altered alpha 1-acid glycoprotein species is at least one factor responsible for decreased tissue-plasma concentration ratios of DMI in PB-treated rats and constitutes an unusual type of drug interaction.