Glial cells exhibit distinct cellular domains: Soma and filopodium(a). Thus, the cytoplasmic pH (pH^cyt) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironment. To address these issues we loaded C6 glial cells with SNARF-1 and evaluated pH^cyt using spectral imaging microscopy. This approach allows us to study pH^cyt in discrete cellular domains with high temporal, spatial and spectral resolution. Because there are differences in cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the soma and filopodium(a). The in situ titration parameters pK_a^', R_max, and R_min had a mean coefficient of variation (MCV) ~6 times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pH^cyt of each region. These studies indicated that glial cells exhibit pH^cyt heterogeneities and pH^cyt , both in the absence and presence of physiological HCO₃^-. The amplitude and frequency of the pH^cyt oscillations were affected by alkalosis, acidosis and by inhibitors of the ubiquitous Na⁺/H⁺ exchanger and HCO₃^--based H⁺-transporting mechanisms. Optical imaging approaches in conjunction with BCECF as a pH probe corroborated the existence of pH^cyt oscillations in glial cells.