The dragline fiber of spiders is composed of two proteins, the major ampullate spidroins I and II (MaSpI and MaSpII). To better understand the assembly mechanism and the properties of these proteins, the adsorption behavior of the recombinant proteins of the spider Nephila clavipes produced by Nexia Biotechnologies Inc. has been studied at the air-water interface using ellipsometry, surface pressure, rheological, and infrared measurements. The results show that the adsorption is more rapid and more molecules are present at the interface for MaSpII than for MaSpI. MaSpII has thus a higher affinity for the interface than MaSpI, which is consistent with its higher aggregation propensity in water. The films formed at the interface consist of networks containing a high content of intermolecular beta-sheets as revealed by the in situ polarization modulation infrared absorption reflection spectra. The infrared results further demonstrate that, for MaSpI, the beta-sheets are formed as soon as the proteins adsorb to the interface while for MaSpII the beta-sheet formation occurs more slowly. The amount of beta-sheets is lower for MaSpII than for MaSpI, most likely due to the presence of proline residues in its sequence. Both proteins form elastic films, but they are heterogeneous for MaSpI and homogeneous for MaSpII most probably as a result of a more ordered and slower aggregation process for MaSpII. This difference in their mechanism of assembly and interfacial behaviors does not seem to arise from their overall hydrophobicity or from a specific pattern of hydrophobicity, but rather from the longer polyalanine motifs, lower glycine content, and higher proline content of MaSpII. The propensity of both spidroins to form beta-sheets, especially the polyalanine blocks, suggests the participation of both proteins in the silk's beta-sheet crystallites.