Basement membrane promotes the reassembly of isolated type II alveolar cells into alveoli-like structures, a process attributable in part to a novel cell adhesion site in the alpha 1-chain of laminin-1 (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The possibility that basement membrane contains other alveolarization activities was probed by subtraction analysis and use of neutralizing antibodies. Deletion of components < 100 kDa, and subsequently < 10 kDa, reduced alveolar cross-sectional area by 70% to 22-25 x 10(3) microns2: the approximate size of alveolar-like structures formed on purified laminin-1 alone. The deleted basement membrane material was adhesive for type II alveolar cells but failed to support alveolar formation in the absence of laminin-1. Preincubation of basement membrane with neutralizing anti-epidermal growth factor (EGF), -basic fibroblast growth factor (bFGF), -insulin-like growth factor (IGF)II, or -transforming growth factor (TGF)-beta antibodies had no inhibitory effect. Because both subtracted basement membrane preparations have in common the exclusion of components < 10 kDa, these results are interpreted as pointing to a sub-10-kDa alveolarization activity(s) that plays a key accessory role in laminin-1-dependent alveolar formation.