In striated muscle, the plasma membrane forms tubular invaginations (transverse tubules or T-tubules) that function in depolarization-contraction coupling. Caveolin-3 and amphiphysin were implicated in their biogenesis. Amphiphysin isoforms have a putative role in membrane deformation at endocytic sites. An isoform of amphiphysin 2 concentrated at T-tubules induced tubular plasma membrane invaginations when expressed in nonmuscle cells. This property required exon 10, a phosphoinositide-binding module. In developing myotubes, amphiphysin 2 and caveolin-3 segregated in tubular and vesicular portions of the T-tubule system, respectively. These findings support a role of the bilayer-deforming properties of amphiphysin at T-tubules and, more generally, a physiological role of amphiphysin in membrane deformation.