Recently a variety of electrophoretic techniques have been used to detect variation not apparent under standard conditions of electrophoresis. This additional variation has been found at the highly polymorphic xanthine dehydrogenase, esterase-5, and aldehyde oxidase loci in Drosophila pseudoobscura and D.persimilis. The present study examined five second chromosome loci in D.melanogaster to determine whether further allelic variation could be revealed for these much less polymorphic loci. A sequential electrophoretic survey, using different buffer pH's and polyacrylamide gel concentrations, was performed on the monomorphic malate dehydrogenase (Mdh) and glutamate oxaloacetate transaminase (Got) loci, and the polymorphic pC-glycerophosphate dehydrogenase (p^-Gpdh) , alcohol dehydrogenase (Adh) , and hexokinase (Hex-3) loci. A total of 163 lines isochromosomal for the second chromosome were examined. No additional allelic variation could be detected at any of these loci. In addition, the Adh locus was examined in a series of gel concentrations ranging from 4% to 10%; Ferguson plot parameters for 52 isochromosomal lines were compared by means of 95% confidence ellipses for the Kr-Mo points and also by estimating error envelopes for the Kr-MidY values. No further variation was uncovered. These findings are in contrast to the presence of abundant hidden variation at highly polymorphic loci, and reasons for the lack of variation at these loci are discussed in terms of the structure and function of the enzymes. Certain reservations are expressed regarding the use of Ferguson plot analysis.