Formation of a basal lamina (BL) ensheathing developing skeletal muscle cells is one of the earliest events in mammalian skeletal muscle myogenesis. BL-resident heparan sulfate proteoglycans have been implicated in various processes during myogenesis, including synaptic differentiation. However, attention has focused on the proteoglycan protein core, ignoring the glycosaminoglycan moiety mainly because of a lack of appropriate tools. Recently, we selected a panel of anti-heparan sulfate antibodies applied here to study the spatiotemporal distribution of specific heparan sulfate (HS) epitopes during myogenesis. In mouse intercostal muscle at embryonic day (E14), formation of acetylcholine receptor clusters at synaptic sites coincides with HS deposition. Although some HS epitopes show a general appearance throughout the BL, one epitope preferably clusters at synaptic sites but does so only from E16 onward. During elongation and maturation of primary myotubes, a process preceding secondary myotube development, significant changes in the HS epitope constitution of both synaptic and extrasynaptic BL were observed. As a whole, the data presented here strengthen previous observations on developmental regulation by BL components, and add to the putative roles of specific HS epitopes in myogenesis and synaptogenesis.