Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis.