The oncogenic protein Ski associates with Smad proteins and counteracts their activation of gene expression and growth inhibition in response to transforming growth factor β (TGF-β). Here we show that Ski protein levels are increased in all 44 human melanoma tumor tissues analyzed in vivo. In addition, Ski subcellular localization changes from nuclear, in preinvasive melanomas (melanomas in situ), to nuclear and cytoplasmic in primary invasive and metastatic melanomas. Furthermore, Ski/Smad association in the cytoplasm seems to prevent Smad3 nuclear translocation in response to TGF-β. The biological significance of Ski overexpression in melanomas was established by showing that down-regulation of Ski levels, by antisense Ski vectors, restored TGF-β-mediated growth inhibition. Such inhibition is apparently mediated by up-regulation of the cyclin-dependent kinase-I p21^Waf-1 and inhibition of cyclin-dependent kinase 2 activity. Our results suggest that high levels of Ski in human melanomas produce a disruption of TGF-β signaling phenotypically similar to that in cells harboring mutations in TGF-β receptors or Smad proteins, and this may represent a significant event in the progression of melanomas in vivo.