We performed in ovo nanoinjection of 4-nonylphenol (NP) into embryos of a transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain, which has two genotypic sex markers, and examined the effects on development and sexual differentiation. The transgene consisted of a green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene. Germ cell-specific GFP expression was visualized in the gonad through the transparent body wall of the living fish. The development of each embryo was observed after nanoinjection of 2.0, 10, 50, 125, or 250 ng of NP. NP administration caused significant higher mortality at > or = 50 ng egg(-1) and inhibited embryonic development, including abnormal hatch and swim-up failure in all treatment groups except 10 ng egg(-1) group. However, it did not cause adverse effects on germ cell proliferation by 10d posthatch (dph) or sex differentiation of survivors by 100 dph. We concluded that single-dose in ovo exposure to nonylphenol affected embryonic development in the medaka but not gonadal development by 10 dph or sexual differentiation in adult fish by 100 dph. Although further investigations might be needed to elucidate the usefulness of nanoinjection of embryos of this strain, present study indicated that the nanoinjection model using olvas-GFP/STII-YI strain medaka has potential for use in evaluating the effects of chemicals on early development and sexual differentiation.