Ovarian follicle development is a complex process mediated by interactions between oocytes and surrounding follicular cells. In an ovary, oocytes are ultimately released from Graafian follicles, which develop from antral follicles localized near the surface of an ovary. To examine the molecular interaction between these 2 cell types, direct gene transfer to follicular cells as well as oocytes appears to be a promising approach, but few studies have applied this technique. The aim of the present study was to develop a technique for gene transfer to antral follicle cells based on their accessibility near the surface of an ovary. B6C3F1 (a hybrid between C57BL6/N and C3H/HeN) female mice aged 4 or 8 w were anesthesized and their ovaries were exposed. About 100 nl of a solution containing reporter plasmid DNA (0.5 µg/μl) and 0.1% trypan blue was injected into a follicle using a glass micropipette attached to the mouthpiece. A total of 6 follicles were injected per ovary. After injection, the ovary was immediately subjected to in vivo electroporation (EP) using an electroporator with 8 square electric pulses of 50 ms and 50 V. After 24 h, the treated ovaries were excised to examine the expression of reporter constructs by histochemistry. All the injected follicles expressed reporter genes to different extents. Inspection of cryostat sections of ovaries injected with the lacZ expression plasmid demonstrated that 50-100% of follicular cells within a follicle were successfully transfected. However, there were no oocytes within the antral follicles that were negative for such staining (15 follicles tested). Similar results were obtained when the enhanced green fluorescent protein expression plasmid was introduced. The present method based on in vivo EP was found to be very effective for transfection of follicular cells. This approach might be useful to explore the roles of genes related to oogenesis/folliculogenesis, and for reproductive manipulation targeted to antral follicles.