Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-b (TGF-b) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-b-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-b-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiationcompetent cells by using improved antisense molecules. We found that TGF-b mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-b-induced cell cycle arrest and to induce or suppress endogenous cell cycle regulators. Inhibition of Smad4 expression exhibited a partial effect, whereas inhibition of Smad2 expression had no effect. By gene expression profiling, we identified TGF-b-dependent genes that are differentially regulated by Smad2 and Smad3 under regular growth conditions on a genome-wide scale. We show that Smad2, Smad3 and Smad4 contribute to the regulation of TGF-b responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.