Abstract The secreted gp96 vaccine approach is based on the use of cellular vaccines (293 cells) that continuously secrete gp96-Ig associated with antigenic peptides that are cross presented to CD8 T cells in vivo. Cellular gp96 vaccines are highly active in generation CTL for cancer therapy and SIV prophylaxis. Here, we investigate the use of gp96-Ig cDNA as vaccine platform. An optimized electroporation approach was used to deliver the B45 plasmid encoding secreted gp96-Ig and HIV-gag-gfp or ovalbumin genes. The B45 plasmid is a bovine papilloma virus derived vector from which the potentially transforming genes E5, E6, and E7 have been removed as well as the L1 and L2 viral capsid genes. The plasmid (50ug) was injected into the muscle (musculus tibialis anterior) of C57/Bl6 mice followed by electric field pulse that induced temporary and reversible pores in the muscle cell membrane. 48h after i.m. injection and EP-delivery, we found gfp (HIV-gag-gfp) expression in the muscle cells. To determine gp96-Ig secretion and cross presentation to CD8 cells we used cDNA for ovalbumin as antigen in conjunction with gp96-Ig and expansion of OT-I TCR-transgenic cells as readout. Vaccination by electroporation increased the frequency of OT-I in the spleen and in the mucosal compartment (lamina propria, small intestine).This simple, efficient and reproducible method of delivery may have great potential for further development of DNA gp96-Ig vaccination.