C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca²⁺-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB₁-EGF-CUB₂ interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.