Recently, it was confirmed that embryos derived from diapausing eggs of the silkworm, Bombyx mori, begin their development and reach larval maturity on mulberry leaves, when the naked eggs are cultured in vitro. In this study, we found that the method of embryo culture is useful for determining the physiological regulation of diapause. We show that the development of embryos derived from diapausing eggs was strongly inhibited by the addition of either sorbitol or trehalose to the culture medium. Furthermore, this inhibitory effect disappeared when the embryos were cultured in a control medium which did not contain either sorbitol or trehalose, indicating that the inhibitory reactions caused by both substances are reversible. The minimal effective dose of either sorbitol or trehalose was approximately 0.2 M, a value similar to the in vivo concentration of sorbitol in diapausing eggs (0.2 M). Glycerol, mannitol or glucose were moderately effective for inhibition. Sorbitol present in diapausing silkworm eggs does not appear to serve as an antifreeze, but as an strong arresting factor of embryonic development. Furthermore, these results show that a decrease in sorbitol releases the embryos from diapause at the termination of diapause.