Recently a novel laminin gamma3 chain was identified in mouse and human and shown to have the same modular structure as the laminin gamma1 chain. We expressed two fragments of the gamma3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The gamma3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the beta2 VI/V fragment. The gamma3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the gamma1 III3-5 fragment. These data suggested that laminins containing the gamma3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against gamma3 VI/V and gamma3 III3-5 showed no cross-reaction with homologous fragments from the gamma1 and gamma2 chains of laminin and allowed the establishment of gamma chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the gamma3 chain compared with the gamma1 chain in various tissue extracts of adult mice. The expression of gamma3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the gamma3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the gamma3 chain to basement membranes of these tissues.