CD9 is a member of the tetraspan (TM4) family of proteins and is abundantly expressed in the epidermis. As CD9 forms complexes with beta 1 integrins and the integrins are known to regulate keratinocyte behaviour, we investigated CD9 expression and function in human epidermal keratinocytes. CD9 was present in all the living layers of the epidermis, whereas the beta 1 integrins were largely confined to the basal layer; the same relative distribution was found in stratified cultures of keratinocytes. There was extensive co-localisation of CD9 and beta 1 integrins on microvilli and at cell-cell borders of basal keratinocytes; however, in contrast to the integrins, CD9 was not found in focal adhesions. CD9 was detected in beta 1 integrin immunoprecipitates and also in immunoprecipitates of CD44 and syndecan, but not of cadherins. CD9 was associated with alpha 3 beta 1 but not alpha 5 beta 1; small amounts of CD9 also co-immunoprecipitated with antibodies to alpha 2 beta 1 and alpha 6 beta 4. Antibodies to CD9 did not affect the proportion of keratinocytes that adhered to laminin 1, type IV collagen and fibronectin, but did inhibit motility of keratinocytes on tissue culture plastic. Like antibodies to the beta 1 integrin subunit, anti-CD9 inhibited suspension-induced terminal differentiation. These results suggest that CD9 may play a role in regulating keartinocyte motility and differentiation.