Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9. Some of the triple helical procollagen IV molecules were associated at their NH2 ends to tetramers which were identified by electron microscopy, velocity sedimentation, and electrophoresis. The formation of these tetramers in cell cultures and from isolated procollagen IV molecules was investigated. After an initial noncovalent association, which is reversible, disulfide bonds form between molecules. Even alkylated molecules form disulfide-linked tetramers when exposed to a mixture of reduced and oxidized glutathione. This reaction requires an adequate concentration of procollagen and is not facilitated by added laminin, Ca2+, or Mg2+ ions. Cystine, as a normal constituent of cell culture media, interferes in tetramer assembly, presumably by forming mixed disulfides. Tetramers formed normally and under the influence of glutathione are similar, but probably not identical, and resemble those isolated from fragmented basement membranes. We conclude that the NH2 ends of procollagen IV molecules can associate into tetramers without the help of other molecules and that disulfide bridges subsequently stabilize the association in various ways.