Uniaxially oriented, piezoelectric silk films are prepared by a two-step method that involves first air drying aqueous, regenerated silk fibroin solutions into films, and then drawing the silk films to a desired draw ratio. The utility of two different drawing techniques-zone drawing and water-immersion drawing-is investigated for processing the silk for piezoelectric studies. Silk films zone drawn to a ratio of lambda = 2.7 display relatively high dynamic shear piezoelectric coefficients of d(14) = -1.5 pC N-1, corresponding to an increase in d(14) of over two orders of magnitude due to film drawing. A strong correlation is observed between the increase in silk II, beta-sheet content with increasing draw ratio as measured by FTIR spectroscopy (C-beta proportional to e(2.5 lambda)), the concomitant increasing degree of orientation of beta-sheet crystals detected via wide-angle X-ray diffraction (full width half maximum (FWHM) = 0.22 degrees for lambda = 2.7), and the improvement in silk piezoelectricity (d(14) proportional to e(2.4 lambda)). Water-immersion drawing leads to a predominantly silk I structure with a low degree of orientation (FWHM = 75 degrees) and a much weaker piezoelectric response compared to zone drawing. Similarly, increasing the beta-sheet crystallinity without inducing crystal alignment, e.g., by methanol treatment, does not result in a significant enhancement of silk piezoelectricity. Overall, a combination of a high degree of silk II, beta-sheet crystallinity and crystalline orientation are prerequisites for a strong piezoelectric effect in silk. Further understanding of the structural origins of silk piezoelectricity provides important options for future biotechnological and biomedical applications of this protein.