Template-assembled synthetic proteins (TASPs) designed to adopt globular, four-helix bundle structures form ion channels in lipid bilayers. The rationale behind this work is that a bundle of amphiphilic alpha-helices may constitute a functional pore-forming motif in a lipid environment. TASPs designated T4(4alpha11), T4(4alpha15), and T4(4alpha18) contain four identical, amphiphilic, helical modules of 11, 15, or 18 residues, respectively; secondary structure modules are attached to the lysine epsilon-amino groups of a cyclic template, Ac-CysLysAlaLysProGlyLysAlaLysCys-NH2, which directs the intramolecular folding. T4(4alpha15) and T4(4alpha18) form cation selective channels with single channel conductances in 500 mM NaCl of 10 and 50 pS for T4(4alpha15) and 9 and 25 pS for T4(4alpha18). In contrast, T4(4alpha11) does not produce discrete conductance events. Notably, channel activity is observed only for molecules that display well-defined alpha-helical structure in solution; K(alpha15), Which contains a single peptide module attached to the epsilon-amino group of Ac-Lys-NH2, does not elicit single channels and displays low alpha-helical content. By contrast, K(alpha18) displays spectral features associated with alpha-helical structure and forms channels with primary conductances of 3 and 9 pS. The occurrence of multiple conductances suggests that molecules aggregate and form heterogeneous conductive oligomers. The minimum length of helical modules required to span a lipid bilayer is established by investigating the channel activity of T4(4alpha15) in bilayers of increasing width. Taken together, results suggest that the relative orientation of amphiphilic segments depends on the hydrophobicity of surrounding media; accordingly, TASP molecules may form ionic channels through a reorientation of template-assembled helical modules to expose charged residues to a central hydrophilic pore.