Cyclohexenyl and cyclohexylidene inhibitors possessing strategically placed olefinic residues, in general, bind to 3-dehydroquinate (DHQ) synthase more tightly than similarly substituted cyclohexyl inhibitors. All of the newly synthesized inhibitors were prepared from a common DHQ derivative. Cyclohexenyl phosphate 1 is the most potent inhibitor of DHQ synthase thus far identified with an inhibition constant (K-i = 1.2 x 10(-10) M), indicating active site binding 1000-fold tighter relative to the corresponding cyclohexyl phosphate 5. Cyclohexenyl tricarboxylate 2 binds 700-fold more tightly than similarly substituted cyclohexyl tricarboxylate 6 and is the first example of a nanomolar-level inhibitor (K-i = 8.6 x 10(-9) M) possessing neither a phosphate monoester nor a phosphonic acid. Cyclohexenyl homophosphonate 4 (K-i = 3.0 x 10(-8) M) and cyclohexylidene homophosphonate 10 (K-i = 3.2 x 10(-9) M) bind 57- and 530-fold, respectively, more tightly than the corresponding cyclohexyl homophosphonate 8. Cyclohexylidene homophosphonate 10 is the first example of a nanomolar-level, homophosphonic acid inhibitor of DHQ synthase. Cyclohexylidene phosphonate 9 (K-i = 2.9 x 10(-10) M) is a 2.9-fold more potent inhibitor relative to cyclohexyl phosphonate 7 which was previously the most potent, slowly-reversible inhibitor of DHQ synthase. Cyclohexenyl phosphonate 3 (K-i = 1.2 x 10(-9) M) is the only olefin-containing, carbocyclic inhibitor where improved binding over the corresponding cyclohexyl analogue was not observed. The impact of olefinic residues in inhibitors on active site binding may indicate that DHQ synthase plays an active catalytic role during E1cb elimination of inorganic phosphate from enzyme-bound substrate.