Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC-25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.