When incubated at 25 degrees C, N-2-grown cells of Frankia strain EAN1pec actively accumulated calcium, while NH4Cl-grown cells did not accumulate calcium. When incubated at 0 degrees C, both N-2-grown and NH4Cl-grown cells did not actively accumulate calcium. Inhibitors of respiration inhibited calcium accumulation by N-2-grown cells at 25 degrees C. Isolated vesicles also accumulated calcium in an energy-and temperature-dependent manner. Two lines of evidence show that Frankia strain EAN1pec has an active calcium extrusion mechanism. First, NH4Cl-grown cells incubated under deenergizing conditions accumulated calcium. Second, calcium efflux from calcium-loaded cells required an energy source and was blocked by inhibitors. The results of this study indicate that Frankia strain EAN1pec has two systems for calcium transport: a calcium extrusion system and a developmentally regulated calcium uptake system.