Extracellular nucleotides cause elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in osteoclasts, although the sources of Ca²⁺ are uncertain. Activation of P2Y receptors causes Ca²⁺ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca²⁺ influx in some cell types. To examine the sources of Ca²⁺, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of [Ca²⁺]_i persisted on removal of extracellular Ca²⁺ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca²⁺-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of [Ca²⁺]_i. After store depletion in the absence of Ca, addition of Ca led to a rise of [Ca²⁺]_i consistent with store-operated Ca²⁺ influx. Store-operated Ca²⁺ influx was greater at negative potentials and was blocked by La³⁺. In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X₄ nucleotide receptors, but with no rise of [Ca²⁺]_i. We conclude that nucleotide-induced elevation of [Ca²⁺]_i in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca²⁺ from intracellular stores.