Calcium cations play a critical role in regulating vesicular transport between different intracellular membrane-bound compartments. The role of calcium in transport between the Golgi cisternae, however, remains unclear. Using a well characterized cell-free intra-Golgi transport assay, we now show that changes in free Ca 2؉ concentration in the physiological range regulate this transport process. The calcium-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid blocked transport with an IC 50 of approximately 0.8 mM. The effect of 1,2-bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid was reversible by addition of fresh cytosol and was irreversible when performed in the presence of a Ca 2؉ ionophore that depletes calcium from lumenal stores. We demonstrate here that intra-Golgi transport is stimulated by low Ca 2؉ concentrations (20 -100 nM) but is inhibited by higher concentrations (above 100 nM). Further, we show that calmodulin antagonists specifically block intraGolgi transport, implying a role for calmodulin in mediating the effect of calcium. Our results suggest that Ca 2؉