The chloroplast plays critical roles in lipid metabolism of microalgae, thus it is recognized as an attractive target of metabolic engineering to enhance biofuel production. It has been well known that recombinant protein expression in microalgal chloroplasts needs specific signal sequence which governs the transition manner of nuclear-encoded polypeptides within the subcellular compartments. However certain microalgae, including diatoms, have complex membrane systems surrounding the chloroplast, and thus chloroplast-targeting protein expression with the signal sequence has rarely been demonstrated except for a few model non-oleaginous diatoms. In this study, we performed recombinant green fluorescence protein (GFP) expression and transportation into the chloroplast of the oleaginous marine diatom, Fistulifera solaris JPCC DA0580. The signal sequence of ATP synthetase gamma subunit, which was predicted to localize in the chloroplast according to a bioinformatics analysis pipeline, was employed as a key factor of this technique. As a result, specific localization of GFP in the chloroplast was observed. It would be useful to engineer the lipid synthesis pathways existing in the chloroplast. Furthermore, intensive gathering of GFP in the rod-like structure was also detected, which has not been observed in model diatom studies. As comparing with electron microscopic observation, the structure was estimated to be a pyrenoid. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.