Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described. While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A. We show that alpha6A-/- mice develop normally and are fertile. The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in alpha6A-/- mice. However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.