This protocol describes the primary culture of individual chromaffin cells derived by enzymatic digestion from the adrenal medulla of the bovine adrenal gland. Since the late 1970s, such cells have provided a useful model system to study neurotransmitter biosynthesis, storage and release in the catecholaminergic system. The protocol can be divided into three stages: isolation of cells (4-6 h), determination of viable cell numbers (approximately 30 min) and growth in culture (3-7 d). An alternative procedure is to perform studies in a continuous chromaffin (pheochromocytoma) cell line, such as PC12, although such transformed cells are typically less highly differentiated than primary cells. The bovine chromaffin cell procedure should yield approximately 10-20 million cells, suitable for several experiments over the subsequent 3-7 d. Typical experiments involve transmitter biosynthesis, vesicular storage, exocytotic release, stimulus coupling (signal transduction) toward secretion or transcription, or morphology, including ultrastructure. The total time, from adrenal gland harvest until functional experiments, is typically 4-8 d.