The secondary structure of Manduca sexta and Sesamia nonagrioides chorion proteins has been studied in intact chorions using laser-Raman and Fourier transform infra-red (FTIR) spectroscopy and in a solution containing extracted and reassembled chorion proteins using circular dichroism (CD) spectroscopy. Laser-Raman and IR spectra suggest the predominance of antiparallel beta-pleated sheet structure in intact chorion proteins of both Lepidoptera species. The bands at 1673, 1674 cm-1 (amide I) and 1234-1238 cm-1 (amide III) in the laser-Raman spectra can best be interpreted as resulting from abundant antiparallel beta-pleated sheet structure. Analysis of the amide I band suggests that chorion proteins consist of 60-70% antiparallel beta-pleated sheet and 30-40% beta-turns. Supporting evidence for the prevalence of antiparallel beta-pleated sheet in chorion proteins was supplied using FTIR spectroscopy by the observation of a very intense absorption band at 1635 cm-1 (amide I) and of a weak band at 1530, 1525 cm-1 (amide II) from chorions of both species. Surprisingly, analysis of the CD spectra of extracted and reassembled chorion proteins suggests that, in solution, they retain a regular secondary structure most probably dominated by beta-pleated sheet. We therefore suggest that the prominent regular beta-sheet structure of chorion proteins may exist in solution and dictate the aggregation and polymerization process in vivo.