The application of electric field pulses in cell suspensions is known to alter membrane integrity, resulting in increased membrane permeabilization. This field-induced membrane poration provides the means to load cells with a variety of external substances, useful for clinical applications. In this work, intact rabbit erythrocytes were successfully loaded with low molecular weight fluorescent probes and with the high molecular weight enzyme pronase, which has been shown to mimic the effects of insulin. Attachment of the enzyme onto the cell surface was also achieved by modifying the applied pulse parameters. Both applications were efficient and accompanied by high cell survival rates. In this way, biological carriers loaded with active substances were produced, offering the potentials for useful clinical applications, either for diagnostic or therapeutic purposes.