The aim of this study was to investigate further the potential of a new hepatocyte culture based on the hypothesis that liver cells in an appropriate in vitro environment (immobilizing gel technique) maintain high metabolic activity comparable with that in vivo. Pimobendan (UD-CG 115), a pyridazinone derivative, is a cardiotonic vasodilator that increases myocardial contractility through calcium sensitization and relaxation of vascular smooth muscle, probably due to phosphodiesterase inhibition. In man, pimobendan is O-demethylated to UD-CG 212. This latter is metabolized to O- and N-glucuronides. Pimobendan itself is also glucuronidated to a N-glucuronide. Human hepatocytes immobilized in collagen gel were incubated with pimobendan to investigate their metabolic activity in the long-term and to compare the results to the data from clinical trials. 14C-labelled pimobendan was incubated at two concentrations (10 and 100 microM) at day 3, 11 and 22 of culture, and samples were analysed after 4, 24 and 48-h incubation. Metabolic patterns were evaluated by hplc with radioactivity-, diode array-, and mass spectral-detection. In vitro, pimobendan was O-demethylated and subsequently O-glucuronidated. The rate of metabolism of pimobendan could be maintained in this culture system for > 3 weeks. However, the relative amount of a putative N-glucuronide under in vitro conditions was lower than in vivo.