A method is described for the preparation of isolated bovine parathyroid cells. Fresh, minced bovine parathyroid tissue is incubated with 2 mg/ml collagenase and 50 mug/ml DNAse for 1 h at 37 C with periodic pipetting. Parthyroid cells are separated from contaminating fat cells and red cells by low speed centrifugation. The resulting cell preparation is indistinguishable from parathyroid cells in intact bovine glands by light and electron microscopy. Hormone release is linear for at least 3 h at both high (2.0 mM) and low (0.5 mM) calcium concentrations and is inversely proportional to divalent cation concentration between 0.3 mM and 1.0 mM calcium as been observed previously both in vivo and in vitro. As in previous studies, hormone release is also stimulated up to 2-fold by 10(6) M (-)isoproterenol, an effect blocked by 10(-6)M (-)propranolol, suggesting a beta-adrenergic effect. Such a cell preparation should be useful for studying hormone binding and effects on cyclic nudleotides and cellular transport phenomena in both normal amd abnormal tissue.