Automatic sequencing of yeast enolase and of its chemically and enzymatically produced peptide fragments has established the sequence of 416 of the 436 residues in the enolase subunits. The missing segments have been provided from results from sequencing the DNA of the yeast enolase genes (Holland, M. J., Holland, J. P., Thill, G. P., and Jackson, K. A. (1981) J. Biol. Chem. 256, 1385-1395). The reported enolase sequence thus represents the results of two completely independent studies, which yielded identical results for 404 of the 436 residues, and which on re-examination are consistent with the reported sequence in all but nine positions. The availability of the entire yeast enolase sequence has permitted a reassessment of structure-function parameters available for the enzyme, and some implications of the sequence information on the secondary, tertiary, and quarternary structure and on the active site components of yeast enolase have been summarized and discussed.