The laminin-nidogen complex, the most abundant noncollagenous component of basement membranes, was recently shown to be a specific substrate for tissue transglutaminase (Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317). Saturation experiments to determine the number of amine acceptor site(s) indicated a single reactive Gln residue in nidogen and none in laminin. Murine nidogen was labeled with [3H]putrescine in the tissue transglutaminase-catalyzed reaction, and two major radioactively labeled fragments, T70 and T40, were isolated after limited trypsin digestion. NH2-terminal sequencing showed that T40 is contained in T70 and corresponds to the rodlike structure of nidogen, made up of epidermal growth factor-like repeats. Three radioactively labeled peptides, obtained by extensive trypsin digestion of reduced and alkylated T40, were sequenced. In all a single residue, Gln726, was found to contain label. Sequencing of additional peptides, obtained after further treatment of the largest radioactively labeled peptide with endoproteinase Asp-N, gave the same result. Gln726 is located in an exposed loop between the second and the third EGF-like repeat in nidogen. This site is also conserved in the human sequence.