The barrier function of plasma membrane in nsPEF-exposed mammalian cells was examined using whole-cell patch-clamp techniques. A specialized setup for nsPEF exposure of individual cells in culture was developed and characterized for artifact-free compatibility with the patch-clamp method. For the first time, our study provides experimental evidence that even a single 60-ns pulse at 12 kV/cm can cause a profound and long-lasting (minutes) reduction of the cell membrane resistance (Rm), accompanied by the loss of the membrane potential. Rm measured in GH3, PC-12, and Jurkat cells (but not in HeLa cells) in 80–120 s after nsPEF exposure was decreased about threefold, and its gradual recovery could take 15 min. Multiple pulses enhanced permeabilization, for example, Rm in GH3 cells fell about 10-fold after a train of five pulses. Within studied limits, permeabilization did not depend on the presence of Ca2+, Mg2+, K+, Cs+, Cd2+, EGTA, tetraethylammonium, or 4-aminopyridine in the pipette or bath solutions. Our results supported theoretical model predictions of plasma membrane poration by nsPEF. However, the extended decrease in Rm, assumed to be related to the life span of the pores, and different nsPEF sensitivity of individual cell lines have yet to be explained. The phenomenon of long-lived membrane permeabilization provides new insights on the nature of nsPEF-opened conductance pores and on molecular mechanisms that underlie nsPEF bioeffects. Bioelectromagnetics 28:655–663, 2007. © 2007 Wiley-Liss, Inc.