Inosine, an endogenous nucleoside, has recently been shown to exert potent effects on the immune, neural, and cardiovascular systems.This work addresses the modulation of intermediary metabolism by inosine through adenosine receptors in isolated rat hepatocytes. We conducted an in silico search in the GenBank and complete genomic sequence databases for additional adenosine/inosine receptors, and for a feasible physiologic role of inosine in homeostasis. Inosine stimulated glycogenolysis (~40%, EC₅₀ 4.2x10^-9 M), gluconeogenesis (~40%, EC₅₀ 7.8x10^-9 M), and ureagenesis (~130%, EC₅₀ 7.0x10^-8 M) compared with basal values; these effects were blunted by the selective A₃ adenosine receptor (AR) antagonist MRS-1220, but not by selective A₁, A_2A, and A_2B AR antagonists. In addition, MRS-1220 antagonized inosine-induced transient increase (40%) in cytosolic Ca²⁺ and enhanced (90%) glycogen phosphorylase activity. Inosine-induced Ca²⁺ mobilization was desensitized by adenosine, in a reciprocal manner inosine desensitized adenosine action. Inosine decreased the cAMP pool in hepatocytes when A₁, A_2A, and A_2B AR were blocked by a mixture of selective antagonists. Inosine-promoted metabolic changes were unrelated with cAMP decrease, but were Ca²⁺-dependent because they were absent in hepatocytes incubated in EGTA- or BAPTA-AM-supplemented Ca²⁺-free medium. After in silico analysis, no additional cognate adenosine/inosine receptors were found in human, mouse, and rat. In both perfused rat liver and in isolated hepatocytes, hypoxia/re-oxygenation produced an increase in inosine, adenosine and glucose release; these actions were quantitatively greater in perfused rat liver that in isolated cells; moreover, all these effects were impaired by the antagonist MRS-1220. Based on results obtained, the known higher extracellular inosine levels under ischemic conditions, and inosine's higher sensitivity for stimulating hepatic gluconeogenesis, it is suggested that after tissular ischemia, inosine contributes to maintain homeostasis by releasing glucose from the liver through stimulation of A₃ adenosine receptors.