Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3-/- mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1-/- mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.