Triple-stranded laminin molecules assemble via an alpha-helical coiled-coil structure spanning approximately 600 amino acid residues of each chain. We reported that the C termini of the beta 1 and gamma 1 chains direct the specific dimer and trimer assembly (Utani, A., Nomizu, M., Timpl, R., Roller, P.P., and Yamada, Y. (1994) J. Biol. Chem. 269, 19167-19175). In this study, we focused on the mechanism of trimer formation of the alpha 2 chain utilizing three different approaches. First, competition assays using mutated recombinant alpha 2 chain defined a 25-amino acid sequence at the C terminus of the long arm as an essential site for assembly with beta 1 and gamma 1 chain. Site-specific mutations and synthetic peptides of this site revealed that both positively charged amino acid residues and the alpha-helical structure within this site were critical. Second, overexpression studies of recombinant alpha 2 chain long arm confirmed that the C-terminal end was critical for the trimer assembly within NIH 3T3 cells. Third, circular dichroism spectroscopic examination of the complexes reconstituted in vitro revealed dynamic conformational changes of the alpha 2 and gamma 1 chains in the process of assembly. These studies also revealed that the proper folding of the extreme C terminus of alpha 2 chain was critical for the stability of trimer. From these data, it is concluded that the C terminus of alpha 2 chain long arm is required for the effective initiation of laminin heterotrimer assembly.