Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)