A new crystalline polymorph of Bombyx mori silk, which forms specifically at the air-water interface, has been characterized This new polymorph has a trigonal crystal structure and is distinctly different from the two previously observed silk crystal structures, silks I and II. Our identification of this new silk polymorph is based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. Electron diffraction indicates that the crystal structure has a trigonal unit cell. This structure consists of a hexagonal packing of chains, each of which assumes a three-fold helical conformation. The resulting crystal structure is found to be similar to that observed for polyglycine II. The sterics of the alanine and serine residues in the crystallizable segments of silk fibroin strongly favor a left-handed 3/2 helix over a right-handed 3/1 helix. Electron diffraction from unoriented samples (powder-type diffraction) provides quantitative support for a left-handed polyglycine II type of 3/2 helical conformation for the silk chains in the crystals of this new polymorph. Single-crystal diffraction patterns and patterns from uniaxially oriented samples are consistent with the proposed crystal structure.