A variety of studies demonstrated a crucial role of mitochondria for clearance of Ca2+ loads in motoneurons. However, previous reports rarely addressed the potential influence of cell dialysis during patch-clamp recordings or temperature on mitochondrial processes. We therefore developed a protocol allowing investigation of Ca2+ dynamics in "undisturbed" AM-ester loaded hypoglossal motoneurons in a slice preparation. By comparing our findings to previous results, we argue against a significant disturbance of mitochondrial buffering by cell dialysis. By varying bath temperatures between 19 and 32 degrees C, we show that temperature alters the rate of mitochondrial uptake but not the relative contribution to maintenance of Ca2+ homeostasis. The results further indicate that mitochondria in hypoglossal motoneurons participate in intracellular Ca2+ regulation at concentrations much lower than has been generally observed for other neurons or neuroendocrine cells. Taken together, our findings further support the important role of mitochondria as regulators of Ca2+ homeostasis in motoneurons.