The epimeric specificity of the catalytic site of rabbit muscle phosphofructokinase was investigated by testing three ketose phosphates as alternate substrates. These (and their epimeric carbons) included: D-psicose-6-P (C-3), D-tagatose-6-P (C-4), and L-sorbose-6-P (C-5). The Michaelis constants (and relative maximal velocities) were: 3.0 mM (45%), 0.054 mM (104%), and 11 mM (15%), respectively. Under the same conditions, D-fructose-6-P had a Km of 0.043 mM and an arbitrary Vmax of 100%. The low affinity of the enzyme for D-psicose-6-P indicates that the L configuration at C-3 is required for effective binding, a specificity similar to several other fructose-metabolizing enzymes. The D configuration at C-5 is also important for tight binding and the proper orientation of the phosphate group of the substrate. The kinetic constants of D-tagatose-6-P were identical with those of D-fructose-6-P, within experimental error. Thus, the configuration at C-4 is not essential for activity; an indication that D-tagatose may be utilized in mammalian tissues. A novel method for the synthesis of D-psicose-6-P and an improved procedure for the synthesis of D-tagatose-6-P are described. All products and intermediates were characterized unequivocally by chemical and physical methods.