The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.