We have found that in mice carrying Pk-3r, an allele leading to loss or activity of kidney pyruvate kinase, the activity of heart pyruvate kinase is also diminished. Electrophoretic studies on tissues from mice carrying Pk-3r and/or Pk-3b, an allele determining an electrophoretically detectable variant, show that Pk-3 affects the expression of both the heart, M1, and the kidney, M2, pyruvate kinase isozymes. These results, together with linkage data, indicate that both isozymes are determined by the same structural gene, Pk-3. We also report a simple method for separating phosphoglucomutase-3 (PGM-3) by electrophoresis on cellulose acetate plates.