Intracellular pH (pH_i) is a powerful modulator of cell excitability (Xiong et al., 2000) yet there is no information on the resting pH_i in dendritic regions. Understanding how dendritic pH_i is regulated is important since epilepsy and some forms of ataxia appear to involve pH_i shifts. We have used fluorescence confocal imaging to investigate regional resting pH_i in Purkinje neurones. Purkinje neurones, in cerebellar slices, were whole-cell patch clamped using pipettes containing a standard solution (Willoughby & Schwiening, 2002) with the fluorescent pH indicator 8-hydroxypyryne-1,3,6-trisulphonic acid (HPTS, 125 μM) and the calcium indicator Fura Red (62.5 μM) at pH 7.3. They were illuminated with alternating 405 nm and 488 nm light and pH-sensitive emission was collected at 505-550 nm and calcium-sensitive emission at 610-700 nm using a Leica SP5 confocal microscope (40x 0.8 NA water immersion objective). The HPTS ratio was calibrated in vitro (Willoughby et al., 1998) using a single-point normalization to the fluorescence of the patch-pipette. Slices were maintained in HEPES-buffered Ringer’s solution. The soma was always more acidic (pH 6.98±0.06 mean±SD) than the dendrites (pH 7.27±0.14 mean±SD at ~120 µm from the soma, n=7) or the patch-pipette (pH 7.3) ~3 mins after the last depolarization (1 s to ~ +20 mV). Further increasing the pH of the patch-pipette to pH 7.6 did not significantly alkalinize the soma. This is not consistent with the pH_i gradient resulting from simple dialysis with the patch-pipette. We considered several other artefacts that might underlie the apparent pH_i gradient including non-linearities in the PMT gain, chromatic aberration and anoxia. To overcome chromatic aberration we produced pH and calcium images from serial confocal sections using only in focus structures. These images showed little systematic relationship between regional pH and either the depth in slice or Ca²⁺ level. However, regional pH and the rate of regulation was related to distance from the soma. Our study suggests a heterogeneity of resting pH and evoked pH shifts in cerebellar Purkinje neurones. It is consistent with most of the functional pH regulation occurring within the dendritic tree rather than at the soma. This could result in the uncoupling of excitability of soma and dendritic arbor and may explain the different reversal potentials reported for GABA_A receptors in different cellular regions.