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Electromechanical coupling, a phenomenon present in collagenous materials, may influence cell-extracellular matrix interactions. Here, electromechanical coupling has been investigated via piezoresponse force microscopy in transparent and opaque membranes consisting of helical-like arrays of aligned type I collagen fibrils self-assembled from acidic solution. Using atomic force microscopy, the transparent membrane was determined to contain fibrils having an average diameter of 76 ± 14 nm, whereas the opaque membrane comprised fibrils with an average diameter of 391 ± 99 nm. As the acidity of the membranes must be neutralized before they can serve as cell culture substrates, the structure and piezoelectric properties of the membranes were measured under ambient conditions before and after the neutralization process. A crimp structure (1.59 ± 0.37 µm in width) perpendicular to the fibril alignment became apparent in the transparent membrane when the pH was adjusted from acidic (pH = 2.5) to neutral (pH = 7) conditions. In addition, a 1.35-fold increase was observed in the amplitude of the shear piezoelectricity of the transparent membrane. The structure and piezoelectric properties of the opaque membrane were not significantly affected by the neutralization process. The results highlight the presence of an additional translational order in the transparent membrane in the direction perpendicular to the fibril alignment. The piezoelectric response of both membrane types was found to be an order of magnitude lower than that of collagen fibrils in rat tail tendon. This reduced response is attributed to less-ordered molecular assembly than is present in D-periodic collagen fibrils, as evidenced by the absence of D-periodicity in the membranes., (Copyright © 2013 Wiley Periodicals, Inc.)