Nucleotide receptors signaling affects cell proliferation, with possible implications on tumorigenic processes. However, molecular targets and action mechanisms of the extracellular nucleotides are still poorly elucidated. We have previously shown in ARO cells that APE1/Ref-1, a transcriptional coactivator responsible for the maintenance of the cellular proliferative rate, is functionally controlled by P2-mediated signaling. Here, we demonstrate that extracellular ATP has a mitogenic effect on ARO cells, increasing ERK phosphorylation, AP1 activation, and cyclin D1 expression. Using the ATP/ADPase apyrase and the P2 receptor antagonist suramin, we show that the extracellular ATP, physiologically released by ARO cells, exerts mitogenic effects. A differential proteomic approach was used to identify molecular events associated with the ATP-induced cell proliferation. Among other proteins, Hsp90 was found upregulated upon ATP stimulation. Pretreatment with suramin completely blocked the ATP-induced Hsp90 activation, confirming the involvement of cell-surface P2 nucleotide receptors in the ATP-mediated activation of ARO cells. Treatment of proliferating ARO cells with suramin and apyrase significantly reduced the intracellular levels of Hsp90, suggesting an autocrine/paracrine mechanism of control on Hsp90 expression by extracellular ATP. The influence of Hsp90 on ATP-induced cell proliferation was also demonstrated by its specific inhibition with 17-AAG. The molecular pathway by which ATP stimulates cell proliferation was further investigated by siRNA strategies showing that Hsp90 is a target of APE1/Ref-1 functional activation. Stimulation of ARO cells with specific nucleotide receptors agonists evidenced a major involvement of P2Y1 and P2Y2 receptors in controlling the Hsp90 activation. Accordingly, these two receptors resulted significantly upregulated in sample biopsies from different thyroid tumors. J. Cell. Physiol. 209: 44–55, 2006. © 2006 Wiley-Liss, Inc.