Transforming growth factor-beta (TGF-beta) signaling in Mv1Lu lung epithelial cells requires coexpression of TGF-beta receptors I (T beta R-I) and II (T beta R-II), two distantly related transmembrane serine/threonine kinases that form a heteromeric complex upon ligand binding. Here, we examine the formation of TGF-beta receptor homo-oligomers and their possible contribution to signaling. T beta R-I can contact ligand bound to T beta R-II, but not ligand free in the medium, and thus cannot form ligand-induced homo-oligomers. T beta R-II, which binds ligand on its own, formed oligomeric complexes when overexpressed in transfected COS cells. However, these complexes were largely ligand-independent and involved immature receptor protein. Since ligand-induced homo-oligomers could not be obtained with the wild-type TGF-beta receptors, we studied receptor cytoplasmic domain homo-oligomerization by using receptor chimeras. The extracellular domain of T beta R-II was fused to the transmembrane and cytoplasmic domains of T beta R-I, yielding T beta R-II/I, and the extracellular domain of T beta R-I was fused to the transmembrane and cytoplasmic domains of T beta R-II, yielding T beta R-I/II. When contransfected with wild-type receptors and exposed to ligand, T beta R-II/I formed a complex with T beta R-I, and T beta R-I/II formed a complex with T beta R-II, thus yielding complexes with homologous cytoplasmic domains. T beta R-II/I transfected alone or with T beta R-I did not restore TGF-beta responsiveness in T beta R-II-defective cell mutants. Furthermore, T beta R-II/I acted in a dominant negative fashion, inhibiting restoration of TGF-beta responsiveness by a cotransfected T beta R-II in T beta R-II-defective cells and by a cotransfected T beta R-I in T beta R-I-defective cells. Similarly, T beta R-I/II transfected alone or with T beta R-II did not restore TGF-beta responsiveness and acted in a dominant negative fashion against T beta R-I. Together with previous genetic and biochemical evidence, these results suggest that TGF-beta mediates transcriptional and antiproliferative responses through the heteromeric T beta R-I.T beta R-II complex and not through homo-oligomeric T beta R-I or T beta R-II complexes.