IGF-I, insulin, FGF-2 and FGF-4 have been implicated in the reciprocal interactions between the apical ectodermal ridge (AER) and underlying mesoderm required for outgrowth and patterning of the developing limb. To study further the roles of these growth factors in limb outgrowth, we have examined their effects on the in vitro morphogenesis of limb buds of the amelic mutant chick embryos wingless (wl) and limbless (ll). Limb buds of wl and ll mutant embryos form at the proper time in development, but fail to undergo further outgrowth and subsequently degenerate. Wl and ll limb buds lack thickened AERs capable of promoting limb outgrowth, and their thin apical ectoderms fail to express the homeobox-containing gene Msx-2, which is highly expressed by normal AERs and has been implicated in regulating AER activity. Here we report that exogenous IGF-I and insulin, and, to a lesser extent, FGF-2 and FGF-4 induce the proliferation and directed outgrowth of explanted wl and ll mutant limb buds, which in vitro, like in vivo, normally fail to undergo outgrowth and degenerate. IGF-I and insulin, but not FGFs, also cause the thin apical ectoderms of wl and ll limb buds to thicken and form structures that grossly resemble normal AERs and, moreover, induce high level expression of Msx-2 in these thickened AER-like structures. Neither IGF-I, insulin nor FGFs induce expression of the homeobox-containing gene Msx-1 in the subapical mesoderm of wl or ll limb buds, although FGFs, but not IGF-I or insulin, maintain Msx-1 expression in normal (non-mutant) limb bud explants lacking an AER. The implications of these results to the relationships among the wl and ll genes, IGF-I/insulin, FGFs, Msx-2 and Msx-1 in the regulation of limb outgrowth is discussed.