Although it is well known that flounder form external asymmetry by migration of one eye at metamorphosis, the control system that forms this asymmetry is unknown. To help elucidate this mechanism, we here describe the embryogenesis and expression profiles of the Nodal-pathway genes in the Japanese flounder, Paralichthys olivaceus. We also perform a comparative study of the laterality of the expression of these genes in sinistral (P. olivaceus) and dextral (Verasper variegatus) flounders. In P. olivaceus, Kupffer's vesicle forms at the 2-somite stage, after which left-sided expression of spaw starts at the 8-somite stage. Left-sided expression of pitx2 occurs in the gut field at the 15-somite to high-pec stages, in the heart field at the 21-somite stage, and in the dorsal diencephalon at the 27-somite to high-pec stages. In response to left-sided pitx2 expression, the heart, gut, and diencephalon begin asymmetric organogenesis at the pharyngula (heart) and the long-pec (gut and diencephalon) stages, whereas the eyes do not show signs of asymmetry at these stages. In both sinistral and dextral flounders, the Nodal-pathway genes are expressed at the left side of the dorsal diencephalon and left lateral-plate mesoderm. Considering these data together with our previous finding that reversal of eye laterality occurs to some extent in the P. olivaceus mutant reversed, in which embryonic pitx2 expression is randomized, we propose that although the Nodal pathway seems to function to fix eye laterality, embryonic expression of these genes does not act as a direct positional cue for eye laterality.