The interaction of rabbit skeletal muscle enolase and 3-phosphoglycerate mutase was detected by an ELISA test, a batch gel-filtration technique, and fluorescence anisotropy measurements, and the activity of enolase was determined to be a function of mutase concentration. The apparent dissociation constant of this enzyme complex is approximately 1 microM. This value seems to be independent of the presence (in fluorescence anisotropy measurements) or the absence (in activity as well as in ELISA experiments) of fluorescein isothiocyanate used widely as a label for determining the complex formation between enzymes in fluorescence anisotropy measurements.