In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin β1 and γ1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal β1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin γ1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns.