Smad family members are newly identified essential intracellular signalling components of the transforming growth factor‐β (TGF‐β) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF‐β signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF‐β or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase‐deficient TGF‐β type I receptor (TβR)‐I after it was phosphorylated by TβR‐II kinase. TGF‐β1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF‐β1 stimulation. Similar results were obtained using HSC4 cells, which are also growth‐inhibited by TGF‐β. Smads 2 and 3 interacted with Smad4 after TβR activation in transfected COS cells. In addition, we observed TβR‐activation‐dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF‐β1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF‐β‐inducible plasminogen activator inhibitor‐1 promoter. Dominant‐negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF‐β induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF‐β signal transduction.