The porinACj is an alpha-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic alpha-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large a helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.