A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffin cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidly than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded approximately 1-2 x 10(8) chromaffin cells per adrenal medulla at a purity of 95% or higher. On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolayer) cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells in suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Very few neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as there was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significantly with time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.