During vertebrate embryonic development, a key to unraveling specific functions of gene products is the capability to manipulate expression of the gene of interest at the desired time and place. For this, we developed a 'microelectroporation' technique by which DNA can be locally introduced into a targeted site of avian embryos, restricting spatial expression of the protein products during development. This technique involved injection of DNA solution in ovo around the target tissue and pinpoint application of an electric field by tungsten electrodes, allowing efficient and reproducible targeted gene transfer, for example, into an optic vesicle, somites, cranial mesoderm and limb mesenchyme. Because of the locality of gene introduction and its expression, survival rates of the embryos were high: approximately 90% of the embryos injected in optic vesicles were alive for at least 1 day after microelectroporation. The instantaneous gene transfer into embryonic cells allowed rapid expression of protein products such as green fluorescence protein within 2.5 h with fluorescence maintained for 3 days of incubation. This improved technique provides a convenient and efficient way to express transgenes in a spatially and temporally restricted manner in chicken embryos.