The purpose of the study was to estimate the genetic effect for skeletal muscle characteristics using pairs of nontwin brothers (n = 32), dizygotic (DZ) twins (n = 26), and monozygotic (MZ) twins (n = 35). They were submitted to a needle biopsy of the vastus lateralis for the determination of fiber type distribution (I, IIa, IIb) and the following enzymes were assayed for maximal activity: creatine kinase, hexokinase, phosphofructokinase (PFK), lactate dehydrogenase, malate dehydrogenase, 3-hydroxyacyl CoA dehydrogenase, and oxoglutarate dehydrogenase (OGDH). For the percentage of type I fibers, intraclass correlations were 0.33 (p < 0.05), 0.52 (p < 0.01), and 0.55 (p < 0.01) in brothers and DZ and MZ twins, respectively. MZ twins exhibited significant within-pair resemblance for all enzyme activities (0.30 ≤ r ≤ 0.68). In spite of these correlations, genetic analyses performed with the twin data alone indicated that there was no significant genetic effect for muscle fiber type I, IIa, and IIb distribution and fiber areas. Although there were significant correlations in MZ twins for all muscle enzyme activities, the often nonsignificant intraclass coefficients found in brothers and DZ twins suggest that variations in enzyme activities are highly related to common environmental conditions and nongenetic factors. However, genetic factors appear to be involved in the variation of regulatory enzymes of the glycolytic (PFK) and citric acid cycle (OGDH) pathways and in the variation of the oxidative to glycolytic activity ratio (PFK/OGDH ratio). Data show that these genetic effects reach only about 25–50% of the total phenotypic variation when data are adjusted for age and sex differences.