This study examined the effect of genotype on prenatal muscle development in both normal-muscled (NM) animals and in double-muscled (DM) animals harboring a mutation in the gene for myostatin that results in the production of a functionally inactive protein. The following muscle development parameters were analyzed at four gestational ages: muscle weight, fiber type, by both enzyme histochemistry and myosin heavy-chain (MHC) immunocytochemistry, and average fiber area. The weights of both M. vastus lateralis and M. vastus medialis were greater throughout prenatal development in the DM animals compared to NM. The percentage of type 1 muscle fibers initially declined with gestational age and subsequently increased in both NM and DM. The percentage of type 1 fibers was consistently lower in DM than in NM. A pattern of MHC isoform localization was shown in DM muscle that is indicative of a delay in muscle development relative to NM. Muscle fiber size was differentially regulated in NM and DM, depending on fiber type. Type 1 fibers were smaller in DM than NM in late gestation, while type 2 fibers were smaller throughout gestation. This study suggests that the inactivating myostatin mutation in DM animals may be associated with changes in both skeletal muscle fiber type and fiber size during bovine muscle development. © 2004 Wiley-Liss, Inc.