Polysialylation of the neural cell adhesion molecule (NCAM) converts it into an anti-adhesive molecule, attenuating intercellular adhesion and repelling apposed membranes. Previous studies have demonstrated that interaxonal repulsion, or defasciculation, induced by polysialylated NCAM (PSA-NCAM) expressed along outgrowing chick motor axons promotes intramuscular branching and facilitates differential guidance of segregating axonal populations. In the present study, we have examined the expression of PSA-NCAM in a developing mammalian motor system during axonal outgrowth, separation of distinct axonal populations, and intramuscular branching. Furthermore, we provide the first clear demonstration of the spatiotemporal modulation of PSA-NCAM expression on myotubes during each stage of myogenesis. Immunohistochemical labelling was used to compare the spatiotemporal pattern of PSA-NCAM expression with those of total-NCAM, the cell adhesion molecule L1, and growth associated protein (GAP-43) during development of the phrenic nerve and diaphragm of fetal rats (embryonic days, E11-E19). During segregation of phrenic and brachial axonal populations at the brachial plexus (E12.5-E13), PSA-NCAM expression was restricted to phrenics, being absent from brachial motoneurons. Both populations labelled equivalently for NCAM, L1, and GAP-43. We postulate that PSA-NCAM may be a component of the molecular machinery that specifically guides phrenic motoneuron growth at the brachial plexus. During diaphragmatic morphogenesis, PSA-NCAM expression: (i) remained high within the phrenic nerve throughout intramuscular branching; (ii) was transiently up-regulated on myotubes during myotube separation associated with primary and secondary myogenesis; (iii) was restricted to those regions of primary and secondary myotube membranes, which were juxtaposed and about to separate. These data suggest a role for PSA-NCAM in the guidance of specific subsets of mammalian motoneurons and in intramuscular branching, and demonstrate an intimate correlation between PSA-NCAM expression and myotube separation.