Abstract In acinar cells of rat pancreas, plasma membrane permeability was investigated after electropulsing (EP). Adenosine 5′-triphosphate (ATP) level was determined to characterize the energy state of the cells. EP ( E = 3 kV cm −1, τ=20 μs) reduced the proportion of trypan blue excluding cells from more than 90% to less than 20%. Thirty minutes after EP, 85% of the cells were found to exclude trypan blue, once again. Immediately after EP, ATP decreased by 15% and oxygen consumption increased by 30%, both indicating an enhanced energy demand. The dependence on energy supply for membrane resealing is demonstrated by the effect of the uncoupler 2,4-dinitrophenol (DNP). DNP added before EP inhibited the membrane resealing in a concentration-dependent manner. In EP-treated cells, DNP led to a reversal of the resealing process when added in the restoration period. The results demonstrate the energy demand of pancreatic acinar cells to maintain the intact state of the plasma membrane. Therefore, the capacity for reversible membrane perforation after EP may be used to evaluate cell viability.