All four of the muscle actins (skeletal, cardiac, vascular, and enteric) in higher vertebrates show distinct expression patterns and display highly conserved amino acid sequences. While it is hypothesized that each of the muscle isoactins is specifically adapted to its respective tissue and that the minor variations among them have developmental and/or physiological relevance, the exact functional and developmental significance of these proteins remains largely unknown. In order to begin to assess these issues, we disrupted the skeletal actin gene by homologous recombination. All mice lacking skeletal actin die in the early neonatal period (day 1 to 9). These null animals appear normal at birth and can breathe, walk, and suckle, but within 4 days, they show a markedly lower body weight than normal littermates and many develop scoliosis. Null mice show a loss of glycogen and reduced brown fat that is consistent with malnutrition leading to death. Newborn skeletal muscles from null mice are similar to those of wild-type mice in size, fiber type, and ultrastructural organization. At birth, both hemizygous and homozygous null animals show an increase in cardiac and vascular actin mRNA in skeletal muscle, with no skeletal actin mRNA present in null mice. Adult hemizygous animals show an increased level of skeletal actin mRNA in hind limb muscle but no overt phenotype. Extensor digitorum longus (EDL) muscle isolated from skeletal-actin-deficient mice at day 2 to 3 showed a marked reduction in force production compared to that of control littermates, and EDL muscle from hemizygous animals displayed an intermediate force generation. Thus, while increases in cardiac and vascular smooth-muscle actin can partially compensate for the lack of skeletal actin in null mice, this is not sufficient to support adequate skeletal muscle growth and/or function.