The binding of 1,N6-ethenoadenosine triphosphate (eATP) to rabbit muscle phosphofructokinase has been studied by fluorescence and circular dichroism, and compared with that of its counterpart, ATP. Muscle phosphofructokinase binds 11.3 +/- 1.2 mol of epsilonATP per tetramer with an average dissociation constant of 60 micrometer. This is in aggreement with the report of 3 ATP binding sites per phosphofructokinase protomer (Kemp & Krebs, 1967). The binding of epsilonATP is relatively homogeneous in comparison with the biphasic binding of ATP. Saturating concentrations of ATP, GTP, and ADP displace about 80% of the bound epsilonATP from the enzyme, whereas FruP2 and AMP displace only 27%. Citrate, on the other hand, enhances the affinity of phosphofructokinase for epsilonATP. The effects of the binding of ATP and epsilonATP on the conformation of enzyme have also been compared. Binding of ATP results in increases in both the lcoal rigidity and the ellipicity of the tryptophanyl side chains, whereas binding of epsilonATP causes a slight decrease in the local rigidity and has virtually no effect on the ellipticity.