A loss of function mutation of the murineα₅ integrin gene generated by gene targeting in embryonic stem cells is a recessive embryonic lethal. The mutant embryos start to show observable defects by day 9 of gestation and die around day 10-11. Theα₅-null embryos have pronounced defects in posterior trunk and yolk sac mesodermal structures, suggesting a role forα₅ β₁ integrin in mesoderm formation, movement or function. However, the embryos progress significantly further than embryos null for fibronectin, for whichα₅ β₁ integrin is a receptor, suggesting the involvement of other fibronectin receptors. In vitro studies on cells derived from theα₅-null embryos confirm that theα₅ β₁ integrin is not expressed on mutant cells and show that the mutant cells are able to assemble fibronectin matrix, form focal contacts, and migrate on fibronectin despite the complete absence of theα₅ β₁ fibronectin receptor integrin. All these functions have previously been thought to involve or requireα₅ β₁. The results presented show that these cellular functions involving fibronectin can proceed using other receptors.