Mouse mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] is a tetrameric protein. Two alleles of the structural gene (Mod-2) are known which code for electrophoretically distinct enzyme subunits: Mod-2a and Mod-2b. A regulatory gene (Mdr-1), closely linked to Mod-2 on chromosome 7, determines the rate of mitochondrial malic enzyme synthesis in brain. Two alleles of Mdr-1 are known: Mdr-1a (high activity) and Mdr-1b (low activitiy). By pulse-labeling with [35S]methionine, immune precipitation, and isoelectric focusing under dissociating conditions, we have measured the relative rates of synthesis of the two types of enzyme subunit in animals of genotypes Mdr-1a Mod-2a/Mdr-1a Mod-2b and Mdr-1S Mod-2a/Mdr-1b Mod-2b. The results show that in the former animals both types of subunit are made at an identical rate, whereas in the latter animals the Mod-2a gene product is synthesized at a rate 2.2 times that of the Mod-2b-coded subunit. Thus we have unambiguously demonstrated that Mdr-1 is cis-active in its control of the expression of the Mod-2 structural gene.