Cells were harvested from four rumen locations in four 2- to 3-yr-old ewes fed fescue hay to determine whether cell origin has an effect on cellular VFA metabolism. Tissue (approximately 150 cm2) was excised from the anterior cranial pillar, ventral sac floor, caudal pillar surface, and dorsal sac ceiling. Cells were isolated using serial tryptic digestion. One milliliter of isolate was incubated for 2 h in 6 mL of medium containing 25 mM propionate and 10 mM butyrate. Incubations were terminated at 0, 30, 60, 90, and 120 min and analyzed for beta-hydroxybutyrate, acetoacetate, lactate, and pyruvate. Cell yield was 22, 22, 24, and 14 (+/- 6) x 106 cells/mL, and viability was 92, 92, 94, and 87% for anterior cranial pillar, ventral sac floor, caudal pillar surface, and dorsal sac ceiling, respectively. All metabolite concentrations and ratios of redox pairs increased throughout the incubations, indicating continuous cellular activity. Final 2-h concentrations (nmol/10(6) cells) were 123, 113, 163, and 158 (+/- 35) for beta-hydroxybutyrate; 38, 42, 24, and 45 (+/- 10) for acetoacetate; 25.3, 20.6, 10.1, and 20.4 (+/- 5.6) for lactate; and 2.54, 0.98, 1.06, and 1.31 (+/- 0.61) for pyruvate in the anterior cranial pillar, ventral sac floor, caudal pillar surface and dorsal sac ceiling incubations, respectively. Origin of rumen tissue had no significant effect on metabolite production, indicating that cellular location is not a critical factor that affects rate of rumen epithelial cell VFA metabolism under these specific in vitro conditions.