The role of motor innervation in supporting and regulating muscle development was studied using aneural muscles in the hindlimb of the mouse mutant peroneal muscular atrophy (pma). This is a single-locus autosomal mutation where homozygous animals lack the common peroneal nerve, so that muscles in the anterolateral compartment of the lower leg develop entirely without innervation. In adults, these muscles are extremely atrophied, and the mice display a clubfoot deformity. The mutant animals provide a preparation in which aspects of muscle formation can be studied in muscles that have never been exposed to direct contact with somatic motor or sensory axons, without pharmacological or surgical intervention. Using quantitative electron microscopy, we found that normal numbers of primary myotubes formed in aneural pma EDL muscles, but a greater than normal proportion degenerated during the first 2 days after their formation. Secondary myotubes appeared at their normal time and position within the muscle, initially in normal numbers, so that the ratio of secondary to primary myotubes initially was greater in pma than in CF1 control strain mice. No abnormalities in ultrastructure were seen until the time of birth, when retardation in development was obvious, together with invading macrophages and degenerating myofibres. The results show that secondary myotube formation in the mouse, as in the chick (B. J. Fredette and L. T. Landmesser, Dev. Biol. 143, 19-35, 1991) is not directly dependent on innervation. In control muscles, secondary myotubes first form in the vicinity of endplates on primary myotubes. No aggregations of ACh receptors or acetylcholinesterase were present in the aneural muscles, showing that these are neurally induced in the mouse, but secondary myotubes formed in their normal position indicating that positional information related to endplate formation is present in aneural muscles.