Electroporation creates transient pores through which exogenous molecules can gain access to the cell cytoplasm. However, the electrical events associated with this phenomenon may perturb membrane-dependent events such as endocytosis. To measure the effect of electroporation on endocytosis, suspensions of human gingival fibroblasts were subjected to 5-ms electrical discharges, allowed to recover for variable periods of time, incubated with fluorescent probes, and then analyzed by flow cytometry. Incubation of electroporated fibroblasts with FITC-conjugated bovine serum albumin (BSA) to label moities on cell membranes nonspecifically demonstrated a time-dependent increase of internalized probe for up to 90 min after electroporation. Pretreatment incubation of cells with cytochalasin D abrogated the increased internalization of FITC-BSA due to electroporation. Compared to controls, fluorescence signals due to internalization of surface glycoproteins with FITC-concanavalin A were 43% higher after electroporation and treatment with endoglycosidase F or H to reduce probe associated with surface membrane. Confocal microscopy confirmed intracellular labeling and reduction of membrane-associated probe by the enzyme. Assessment of nonspecific FITC-Con A labeling of cells by pretreatment with alpha-methyl D-mannoside showed that labeling was largely (92%) specific. Compared to controls, electroporation induced a 60% increase of internalization of lucifer yellow, a fluid-phase endocytosis marker. Dual fluorescence labeling of membrane phosopholipids by FITC and TRITC-DHPE demonstrated an increased acidification after electroporation that was time dependent, indicating that electroporation induced more rapid entry of membrane lipid into endosomal compartments. These data demonstrate that the electrical fields used in electroporation of fibroblasts cause an actin-dependent increase in the internalization of all membrane components examined and an increased rate of probe entry in to acidifying compartments.