The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)–enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenolpyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)–enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.