Sarcolemmal-membrane-associated proteins (SLMAPs) define a new class of coiled-coil tail-anchored membrane proteins generated by alternative splicing mechanisms. An in vivo expression analysis indicated that SLMAPs are present in somites (11 days post-coitum) as well as in fusing myotubes and reside at the level of the sarcoplasmic reticulum and transverse tubules in adult skeletal muscles. Skeletal-muscle myoblasts were found to express a single 5.9 kb transcript, which encodes the full-length ∼91 kDa SLMAP3 isoform. Myoblast differentiation was accompanied by the stable expression of the ∼91 kDa SLMAP protein as well as the appearance of an ∼80 kDa isoform. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Membrane targeting of the de-regulated SLMAPs was not critical for the inhibition of myotube development. Protein–protein interaction assays indicated that SLMAPs are capable of self-assembling, and the de-regulated expression of mutants that were not capable of forming SLMAP homodimers also inhibited myotube formation. These results imply that regulated levels and the temporal induction of SLMAP isoforms are important for normal muscle development.