In addition to being an intermediate along the reaction coordinate catalyzed by myo-inositol l-phosphate (MIP) synthase, myo-2-inosose 1-phosphate is a 3.6 mu M competititve inhibitor of the enzyme at pH 7.2. To probe the importance of the oxidized reaction center in myo-2-inosose l-phosphate to active site interactions and gauge the relative contribution of the keto form to these interactions, three analogues have been examined for MIP synthase inhibition. 2-Deoxy-myo-inositol l-phosphate is used to gauge the impact of removing the oxidized reaction center of myo-2-inosose l-phosphate. 1-Deoxy-1-(phosphonomethyl)-myo-2-inosose, with a methylene group substituted for the phosphate monoester oxygen of myo-2-inosose l-phosphate, exists in neutral aqueous solution exclusively in its keto form. Dihydroxyacetone phosphate provides insights into the minimum set of structural requirements for inhibition of MIP synthase. 2-Deoxy-myo-inositol l-phosphate was a 170 mu M competitive inhibitor at pH 7.2, which is a 47-fold reduction in inhibitor potency relative to myo-2-inosose phosphate. Competitive inhibition of MIP synthase by 1-deoxy-1-(phosphonomethyl)-myo-2-inosose was dependent on solution pH with inhibition constants of 6.4, 37, and 160 mu M measured, respectively, at pH 8.0, 7.2, and 6.4. Dihydroxyacetone phosphate was a 700 mu M competitive inhibitor of MIP synthase at pH 7.2. The measured inhibition constants do not allow MIP synthase inhibition to be unambiguously assigned to the keto form of myo-2-inosose l-phosphate. However, the examined analogues of myo-2-inosose l-phosphate do establish the importance of active site interactions with an oxidized reaction center and suggest that this approach may be a general strategy for inhibiting MIP synthase.