Chronic hypoxia (Chox) augments chemoafferent activity in sensory fibers innervating carotid body (CB) chemoreceptor type I cells, however, the underlying mechanisms are poorly understood. Here, we tested the hypothesis that enhanced paracrine signaling via adenosine (Ado) A2b receptors is involved. Dissociated rat CB cultures were exposed for 24 hr to normoxia (Nox; 21% O₂), chronic hypoxia (Chox; 2% O₂), or the hypoxia mimetic deferoxamine (DFX), and catecholamine (CAT) secretion from type I cells was monitored by amperometry. Compared to Nox controls, Chox and DFX-treated type I cells showed a more robust CAT secretion after acute exposure to acid hypercapnia (10% CO₂; pH 7.1) and high K⁺ (75 mM). Exogenous Ado increased CAT secretion in a dose-dependent manner, and the EC₅₀ was right shifted from ~21 µM Ado in Nox cells to ~78 µM in Chox cells. Ado-evoked secretion in Nox and Chox cells was markedly inhibited by MRS 1754, an A2b receptor blocker, but was unaffected by SCH58261, an A2a receptor blocker. Similarly MRS 1754, but not SCH58261, partially inhibited high K⁺-evoked CAT secretion, suggesting a contribution from paracrine activation of A2b receptors by endogenous Ado. CB chemostimuli, acid hypercapnia and hypoxia, elicited a MRS 1754-sensitive rise in intracellular Ca²⁺ that was more robust in Chox and DFX-treated cells compared to Nox cells. Taken together, these data suggest that paracrine Ado A2b receptor signalling contributes to stimulus-evoked CAT secretion in normoxic and chronically hypoxic CB chemoreceptors; however, the effects of Ado are more robust after chronic hypoxia.