The antimitogenic action of transforming growth factor beta (TGF-beta) in epithelial cells involves cyclin-dependent kinase (cdk) inhibitory gene responses and downregulation of c-Myc expression. Although the cdk inhibitory responses are sufficient for G(1) arrest, enforced expression of c-Myc prevents G(1) arrest by TGF-beta. We investigated the basis of this antagonism by using Mv1Lu lung epithelial cell lines that conditionally express levels of human c-Myc. We show that c-Myc prevents induction of the cdk4 inhibitor p15(Ink4b) and the subsequent inhibition of G(1) cdks by TGF-beta. We assessed the significance of this effect by analyzing the oligomeric state of cdk4 in these cells. In proliferating cells, endogenous cdk4 is distributed among three populations: an abundant high-molecular-mass (>400-kDa) pool of latent cdk4 that serves as a source of cdk4 for cyclin D, a low-abundance pool containing active cyclin D-cdk4 complexes, and an inactive population of monomeric cdk4. Cell stimulation with TGF-beta converts the latent and active cdk4 pools into inactive cdk4, an effect that is specifically mimicked by overexpression of p15 but not by other forms of G(1) arrest. This process of TGF-beta-induced cdk4 inactivation is completely blocked by expression of c-Myc, even though the latent and active cdk4 complexes from c-Myc-expressing cells remain sensitive to dissociation by p15 in vitro. c-Myc causes a small increase in cyclin D levels, but this effect contributes little to the loss of TGF-beta responses in these cells. The evidence suggests that c-Myc interferes with TGF-beta activation of the p15 G(1) arrest pathway. TGF-beta must therefore downregulate c-Myc in order to activate this pathway.