Extracellular nucleotides, signalling through P2 receptors, regulate the function of both osteoblasts and osteoclasts. Osteoblasts are known to express multiple P2 receptor subtypes (P2X2,5,7 and P2Y₁,_2,4,6), levels of which change during differentiation. ATP and UTP potently inhibit bone mineralisation in vitro, an effect mediated, at least in part, via the P2Y₂ receptor. We report here that primary rat osteoblasts express additional, functional P2 receptors (P2X1, P2X3, P2X4, P2X6, P2Y₁₂, P2Y₁₃ and P2Y₁₄). Receptor expression changed with cellular differentiation: e.g., P2X4 receptor mRNA levels were 5-fold higher in mature, bone-forming osteoblasts, relative to immature, proliferating cells. The rank order of expression of P2 receptor mRNAs in mature osteoblasts was P2X4>>P2Y₁>P2X2>P2Y₆>P2X1>P2Y₂>P2Y₄>P2X6>P2X5>P2X7>P2X3>P2Y₁₄>P2Y₁₃>P2Y₁₂. Increased intracellular Ca²⁺ levels following stimulation with P2X-selective agonists indicated the presence of functional receptors. To investigate whether P2X receptors might also regulate bone formation, osteoblasts were cultured for 14days with P2X receptor agonists. The P2X1 and P2X3 receptor agonists, α,β-meATP and β,γ-meATP inhibited bone mineralisation by 70% and 90%, respectively at 1μM, with complete abolition at ≥25μM; collagen production was unaffected. Bz-ATP, a P2X7 receptor agonist, reduced bone mineralisation by 70% and 99% at 10μM and 100μM, respectively. Osteoblast alkaline phosphatase activity was similarly inhibited by these agonists, whilst ecto-nucleotide pyrophosphatase/phosphodiesterase activity was increased. The effects of α,β-meATP and Bz-ATP were attenuated by antagonists selective for the P2X1 and P2X7 receptors, respectively. Our results show that normal osteoblasts express functional P2X receptors and that the P2X1 and P2X7 receptors negatively regulate bone mineralisation.