Chronic activation of proinflammatory caspase-1 in the retinas of diabetic animals and patients in vivo and retinal Müller cells in vitro is well documented. In this study we characterized how elevated glucose and extracellular purines contribute to the activation of caspase-1 in a cultured rat Müller cell (rMC-1) model. The ability of high glucose (25 mM, 24 h) to activate caspase-1 was attenuated by either apyrase, which metabolizes extracellular ATP to AMP, or adenosine deaminase (ADA), which metabolizes extracellular adenosine to inosine. This suggested that autocrine stimulation of ATP-sensing P2 receptors and adenosine-sensing P1 receptors may in part mediate the response to high glucose. Exogenous ATP, 5'-N-ethylcarboxamido-adenosine (NECA), a nonselective P1 receptor agonist, or forskolin (FSK) increased caspase-1 activity in rMC-1 cells cultured in control glucose (5 mM) medium. Accumulation of active caspase-1 was also increased by dipyridamole, which suppresses adenosine reuptake. High-glucose stimulation of caspase-1 was attenuated by suramin, a nonselective P2 antagonist, or A2 adenosine receptor antagonists, but not by antagonism of P2X7 ATP-gated ion channel receptors. Although high glucose increased P2X7 mRNA, neither P2X7 protein nor function was detected in rMC-1 cells. The increased caspase-1 activity stimulated by high glucose, FSK, NECA, or ATP was correlated with increased gene expression of caspase-1 and thioredoxin-interacting-protein (TXNIP). These findings support a novel role for autocrine P1 and P2 purinergic receptors coupled to cAMP signaling cascades and transcriptional induction of caspase-1 in mediating the high-glucose-induced activation of caspase-1 and secretion of IL-1β in a cell culture model of nonhematopoietic retinal Müller cells.