The presynaptic neurotoxin, beta-bungarotoxin, was injected into rat fetuses in utero to destroy the innervation of their hindlimb muscles. These injections were made prior to the invasion of motor axons into the muscles and, in some cases, prior to the cleavage of individual muscles. Examination of the lateral motor column of the spinal cord showed a dramatic reduction (greater than 95%) in the number of motoneuron cell bodies. Staining of sections of the hindlimb with silver and with antibodies to neurofilament proteins and to a synaptic vesicle protein indicated that the muscles were aneural. Anti-myosin antibodies applied to sections of the hindlimb revealed that these aneural muscles by the 20th day of gestation had the same types of fibers as were present in normal muscles of the same age. Moreover, fiber types in most muscles showed their characteristic intramuscular distributions. These findings suggest that fiber types can differentiate in the absence of the nervous system. However, some fibers achieved their ultimate fiber type fate without passing through the normal sequence of myosin expressions. Moreover, some slow fibers lost their slow expression, suggesting that the maintenance of the slow differentiation may require innervation. Muscle growth was dramatically affected by the absence of motoneurons; some muscles were decreased in size and others disappeared completely. In muscles which had not degenerated by the time secondary myogenesis normally begins, secondary muscle fibers were generated indicating that the genesis of these fibers is not strictly nerve dependent. Because fiber types differentiate independently of the nervous system, this study suggests that motoneurons selectively innervate fiber types during normal development.