To clarify the key compounds that account for the umami taste of soy sauce, a typical Japanese soy sauce, Koikuchi Shoyu, was separated by preparative chromatography, and the umami enhancing fractions were screened on the basis of an umami intensity of a 6.0 mM monosodium L-glutamate (MSG) solution. Liquid chromatography-time of flight mass spectrometry (LC-TOFMS), 1D/2D nuclear magnetic resonance spectroscopy (NMR) studies of the umami enhancing fractions led to the identification of N-(1-deoxy-D-fructos-1-yl)pyroglutamic acid (Fru-pGlu), N-(1-deoxy-D-fructos-1-yl)valine (Fru-Val), N-(1-deoxy-D-fructos-1-yl)methionine (Fru-Met), pyroglutamylglutamine (pGlu-Gln), and pyroglutamylglycine (pGlu-Gly). Although all the compounds identified were at sub-threshold concentrations in the soy sauce, a taste reconstitution experiment revealed that they contributed part of the umami taste of the soy sauce.