Extracellular nucleotides acting through P2 receptors elicit a wide range of responses in many cell types. There is increasing evidence that adenosine triphosphate (ATP) may function as an important local messenger in bone and cartilage. In this study, we used immunocytochemistry, employing novel polyclonal antibodies against P2X(1-7) receptors, and in situ hybridization, using oligonucleotide probes corresponding to P2X(2,4) and P2Y(2,4) messenger RNAs (mRNAs), to localize P2 receptors on undecalcified bone sections and on cultured osteoblasts and osteoclasts. We provide the first direct evidence that the P2X(2) receptor subtype is expressed on osteoclasts, osteoblasts, and chondrocytes. We also obtained evidence for the expression of P2X(5) and P2Y(2) receptors on osteoblasts and chondrocytes, and for P2X(4) and P2X(7) receptors on osteoclasts. Our results confirm earlier reports of P2Y(2) and P2X(4) expression in human osteoclastoma and rabbit osteoclasts, respectively, and are consistent with ATP responses observed on bone cells using electrophysiological techniques. Our novel finding that P2X(2) is expressed by osteoclasts is of particular interest. P2X(2) is the only P2 receptor subtype that requires extracellular acidification to show its full sensitivity to ATP, and our recent functional studies have shown that the stimulatory action of ATP on resorption pit formation by mature osteoclasts is amplified greatly at low pH. These findings point to fundamental new mechanisms for the local modulation of bone resorption.