The Cnidarian, Hydra, is a simplified metazoan whose body wall is composed of an epithelial bilayer with an intervening extracellular matrix termed the mesoglea. Hydra mesoglea has been shown to have a number of components seen in higher invertebrate and vertebrate matrices, including fibronectin, type IV collagen, laminin, and heparan sulfate proteoglycan. Based on previous studies which indicated that extracellular matrix components are critical to hydra development, the current study was designed to determine the role of intact fibronectin and its various functional domains during development of hydra cell aggregates. Hydra cell aggregation involves the complete morphogenesis of adult hydra from pellets of dissociated hydra cells. During this development process, cells segregate into an epithelial bilayer and then deposit a new extracellular matrix prior to continuation of morphogenesis. Results from this study demonstrate that intact fibronectin and its 30-kDa gelatin binding domain are effective blockers of hydra cell aggregate development. The gelatin binding capacity of the 30-kDa fragment is lost upon reduction and in the present studies reduction and alkylation of this fragment resulted in a loss in its ability to block hydra cell aggregate development. Because of these findings and the potential for collagen interaction with the 30-kDa gelatin binding domain, studies were also performed with various type IV collagen domains. These studies indicated that both the NC1 and 7 S domains of type IV collagen were also effective blockers of hydra cell aggregate development. These results were mimicked when antibodies to fibronectin or type IV collagen were used. Structural changes in mesoglea, inhibition of cell proliferation, and changes in cell differentiation patterns accompanied the blockage of hydra cell aggregates. These results indicate that blockage may be due to alterations in mesoglea structure with accompanying effects on cell behavior. It is concluded that (i) fibronectin and type IV collagen are critical to the early stages of hydra cell aggregate development when the mesoglea is initially formed and that (ii) perturbation of aggregate development by fragments of these extracellular matrix components results in alterations in hydra cell division, cell differentiation, and morphogenesis.