The factors regulating astroglial resting membrane potential (RMP) are unresolved. Here, we have examined developmental, morphological, and intracellular factors that may regulate the RMP in astrocytes of isolated intact optic nerves of rats and mice aged postnatal day (P3) to adult. The astroglial RMP ranged from −25 to −85 mV, independent of age and morphological phenotype. There was a developmental negative shift in the astroglial RMP from a non-Gaussian distribution in perinatal nerves, to a bimodal distribution of RMPs after P15, with peaks at −52 and −74 mV in adults. Blockade of Kir with 100 μM BaCl2 significantly depolarized the RMP to −30 mV; the RMP was unaffected by TEA or agents that modulated ATP-sensitive potassium channels. Raising intracellular cyclic AMP (cAMP) with dbcAMP or forskolin induced a significant hyperpolarization by ∼15 mV, whereas inhibition of cAMP-dependent protein kinase (PKA) depolarized the astroglial RMP to −40 mV. The hyperpolarizing action of dbcAMP was blocked by 100 μM BaCl2. The effects of BaCl2 indicate that the developmental negative shift in the RMP and the cAMP-mediated hyperpolarization were dependent on Kir. This study provides evidence that the heterogeneous RMP of mature astrocytes is regulated by cAMP and PKA signaling.