Cell aggregates of bovine parathyroid tissue were prepared by limited collagenase digestion and placed in culture in Weymouth's MB752/1 (calcium = 3.3 mg/100 ml) containing 5% fetal bovine serum and supplemented with insulin alone, or insulin, hydrocortisone, transferrin and epidermal growth factor. Only insulin was required for the maintenance of PTH secretion over a 9-day period. The cell aggregates spread to form monolayer in 3-5 days. The majority of the cells in monolayer were polygonal with well-defined borders. Nuclei were round and the cytoplasm was free of vacuoles. Cell cultures responded to secretory stimulation by low calcium or by isoproterenol with increases in the secretion of PTH and SP-1. At low calcium, about 18% of both the cellular PTH and SP-1 was secreted per hour, and up to 50% of the cell content of these proteins was released per hour upon stimulation by isoproterenol and low calcium combined. The responses to calcium and isoproterenol decreased as a function of time in culture, and calcium responses often disappeared completely by 10 days of culture. When cells were cultured in medium containing a higher (5 mg%) than standard concentration of calcium between days 3-6 of culture, the degree of secretory inhibition attainable with high calcium was greater than that of cells cultured in the standard medium. When secreted hormonal peptides were separated by SDS-gel electrophoresis prior to RIA, it was found that the secretion of intact hormone was sensitive to calcium. For every molecule of PTH secreted into the medium, 1.5-2 mole-equivalents of carboxyl fragments were also released. Calcium control of fragment release was not as stringent as that of PTH release.