An enriched preparation of rat hepatocyte couplets was obtained by collagenase perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent). Canalicular secretory activity (the ability to accumulate cholyl-lysyl-fluorescein, CLF) was first apparent after 2 h of culture at 37 degrees C and was present in over 80 per cent of the total population after 5-6 h. This remained almost constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4 degrees C for up to 6 h prior to incubation had no adverse effect upon secretory function. Reduction of canalicular secretory activity occurred at a concentration of the hepatotoxic agent menadione (IC50 17 microM) that was lower than that required to induce mild plasma-membrane blebbing (IC50 43 microM). This study has optimized and characterized the canalicular secretory effectiveness and stability of an enriched preparation of hepatocyte couplets, and established the feasibility of studies of toxic agents on hepatobiliary function in a heterogeneous population of hepatocytes. In this preparation other biochemical parameters can be assessed, thus complementing previous techniques using individual couplets.