Two malic enzyme alleles, Men(113A) and Men(113G), occur at approximately equal frequency in North American populations of Drosophila melanogaster, while only Men(113A) occurs in African populations. We investigated the population genetics, biochemical characteristics, and selective potential of these alleles. Comparable levels of nucleotide polymorphism in both alleles suggest that the Men(113G) allele is not recently derived, but we find no evidence in the DNA sequence data for selection maintaining the polymorphism. Interestingly, the alleles differ in both V(max) and K(m) for the substrate malate. Triglyceride concentration and isocitrate dehydrogenase (IDH) and glucose-6-phosphate dehydrogenase (G6PD) activities are negatively correlated with the in vivo activities of the Men alleles. We examined the causality of the observed correlations using P-element excision-derived knockout alleles of the Men gene and found significant changes in the maximum activities of both IDH and G6PD, but not in triglyceride concentration, suggesting compensatory interactions between MEN, IDH, and G6PD. Additionally, we found significantly higher than expected levels of MEN activity in knockout heterozygotes, which we attribute to transvection effects. The distinct differences in biochemistry and physiology between the naturally occurring alleles and between the engineered alleles suggest the potential for selection on the Men locus.