Interactions between the homeostasis of intracellular ions were studied in astrocytes within perfused, isolated optic nerve of humanely killed 0-2 post-natal day rat nerves. Optic nerves were dissected and astrocytes loaded with one of a pair of fluorescent indicators (either SBFI+BCECF or FURA-FF+BCECF) to compare changes in [Na⁺]_i, [Ca²⁺]_i and pH_i. Images were collected at 0.5 Hz from individual cells using a standard imaging system. [Na⁺]_i and pHi were calibrated using the one-point method of Boyarsky et al 1993. [Ca²⁺]_i changes were not calibrated and changes in 340/380 fluorescence are reported. An initial 5 min period of control recording in HEPES-buffered ACSF was followed by a switch to HCO₃-buffered ACSF. This evoked a DIDS (500 μM) -inhibitable alkalinisation of 0.37 ±0.02 pH units (mean ±SEM). This pH_i change was not significantly effected by removal of [Na⁺]_o (0.29 ±0.03 pH units; P>0.05 using ANOVA), consistent with stimulated acid extrusion via HCO3/Cl^– exchange. An 5-(N-ethyl-N-isopropyl) amiloride (EIPA; 10 μM) -inhibitable rise in [Na⁺]_i of 8.8 ±0.8 mM was evoked by switching from HEPES- to HCO3-aCSF, which is consistent with an increase in acid-extrusion via Na-H exchange. Astrocytes shrank significantly upon exposure to HCO3, assessed by changes in dye concentration (3.8 ±0.1% increase in dye fluorescence after 10 min, n=69). This effect was potentiated by EIPA (10.6 ±0.5% increase in dye fluorescence, n=67; p<0.001) and significantly reduced by DIDS (2.20 ±0.03% increase in dye fluorescence, n=69; p<0.01). Increased acid-extrusion via Na-H exchange in the presence of HCO₃ therefore mitigated cell shrinking that resulted from Cl^– -efflux via HCO₃/Cl^– exchange. [Ca²⁺]_i increased upon switching to HCO₃ (0.080 ±0.003% increase), an effect that was significantly reduced under conditions that inhibit Na-Ca exchange (e.g., a 0.049 ±0.006% increase was seen in 50 μM bepridil; P<0.005). The [Ca²⁺]_i rise was completely blocked by either DIDS or EIPA, indicating a complex interaction involving HCO₃/Cl^– exchange activation by HCO₃, cell shrinking, stimulation of acid-extrusion via Na-H exchange, Na+ influx and inward (Ca²⁺-influx) operation of Na-Ca exchange. These immature neural cells are known to express relatively low levels of Ca²⁺-ATPase and high levels of Na-Ca exchanger, which may result in these marked and complex interactions between membrane ion transporters.