Ever since isozyme–allozyme polymorphisms were detected there has been an incessant effort to relate the amount of observed protein variation to various aspects of molecular structure and function. Structural–functional constraints can limit the amount of overall genie variation, and the observed variation can be due to neutral mutation and random genetic drift, mutation–selection balance, or balancing selection. The present analysis of structural–functional constraints on gene–enzyme variation in natural populations of Drosophila melanogaster shows that while both subunit size and substrate group show significant effects on number of alleles and mean heterozygosity, only substrate group affects population structure significantly. Subunit structure (monomer vs. multimer), IUB enzyme class (e.g., tranferase, lyase), and tolerance of null alleles showed no significant effect on level or pattern of variation. While partly interdependent these results suggest that efforts should be made to distinguish between constraints arising from molecular structure and physiological function.Key words: structural–functional constraints, subunit size, gene-enzyme variation, selection, neutrality.