Critical investigation into α-cell biology in health and diabetes has been sparse and at times inconsistent because of the technical difficulties with employing conventional strategies of isolated islets and dispersed single cells. An acute pancreas slice preparation was developed to overcome the enzymatic and mechanical perturbations inherent in conventional islet cell isolation procedures. This preparation preserves intra-islet cellular communication and islet architecture in their in situ native state. α-Cells within tissue slices were directly assessed by patch pipette and electrophysiologically characterized. The identity of the patched cells was confirmed by biocytin dye labelling and immunocytochemistry. α-Cells in mouse pancreas slices exhibited well-described features of I(Na) (excitable at physiological membrane potential), I(KATP), small cell size, low resting membrane conductance, and inducible low and high voltage-activated I(Ca), the latter correlating with exocytosis determined by capacitance measurements. In contrast to previous reports, our large unbiased sampling of α-cells revealed a wide range distribution of all of these parameters, including the amount of K(ATP) conductance, Na+ and Ca2+ current amplitudes, and capacitance changes induced by a train of depolarization pulses. The proposed pancreas slice preparation in combination with standard patch-clamping technique allowed large sampling and rapid assessment of α-cells, which revealed a wide distribution in α-cell ion channel properties. This specific feature explains the apparent inconsistency of previous reports on these α-cell ion channel properties. Our innovative approach will enable future studies into elucidating islet α-cell dysregulation occurring during diabetes.