ABSTRACT Seven alleles at the esterase-5 locus of Drosophila pseudoobscura appear approximately uniformly spaced on 5% acrylamide gels. Such stepwise "ladders" in mobility have been used to argue for the charge-state model of electrophoretic mobility. To evaluate this interpretation, flies of the seven strains were examined in replicate electrophoresis on polyacrylamide gels of differing pore size, permitting estimation of the relative contributions of charge and of size/conformation to electrophoretic mobility. Six of the seven strains examined proved to be heterogeneous, containing multiple variants that migrate to similar positions on 5% acrylamide gels. In the one strain genetically analyzed to date, the hidden variants segregate in crosses. A total of fourteen variants are detected by this gel sieving analysis, many of them involving apparent conformational differences. Thus, protein properties in addition to net charge appear to play an important role in determining the degree of mobility difference between alleles. Examining estimates of free mobility, uniform charge differences are the rule within conformational classes. However, the superposition of conformational heterogeneity renders interpretation of mobility spacing solely in terms of such charge differences inappropriate.