1. 1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca²⁺ stores in chromaffin cells. 2. 2. The thapsigargin-sensitive compartment of Ca²⁺ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15-30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. 3. A Ca²⁺-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca²⁺-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [γ-³²P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca²⁺ ATPases. 4. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca²⁺ uptake. 5. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca²⁺ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.