Thyroid hormones play a role in the regulation of glutathione S-transferase (GST) expression. Here, co-cultures of rat hepatocytes with bile duct epithelial cells have been used to study the direct effects of both triiodothyronine (T3) and thyroxine (T4) on GST activities and proteins. Because T3 and T4 are poorly water soluble and organic solvents used to dissolve them often interfere with biotransformation pathways, an alternative delivery system namely hydroxypropyl-beta-cyclodextrin (HPBC) has been applied. Appropriate control cultures contained either 0.02 or 0.10% (w/v) HPBC, the concentrations necessary to supply T3 and T4 (10(-9) to 10(-5) M) to the cells, respectively. No effect of the vehicle HPBC on the different GST isoenzyme activities and proteins could be observed. On the contrary, after 10 days of co-culture, T3 and T4 decreased GST protein concentrations as well as GST activities measured by 1-chloro-2,4-dinitrobenzene (broad spectrum), 1,2-dichloro-4-nitrobenzene (Mu class M1/M2-specific) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Alpha class A1/2-specific) in a concentration-dependent manner. The Alpha class subunits A1/2 and A3, and the Mu class subunit M2 were mostly affected. No effect was observed on the Pi class enzyme. These findings indicate that a combination of co-cultured hepatocytes with an HPBC-based delivery system for hydrophobic compounds represents a powerful in vitro tool in drug development.