['Transforming growth factor β (TGF-β) is a potent inhibitor of cell proliferation that regulates cell functions by activating specific serine/threonine kinase receptors on the cell surface. Type I TGF-β receptor (TβR-I) is essential for TGF-β signaling, and substrates of TβR-I provide insights into molecular mechanisms of TGF-β signaling.', 'Here we identify eukaryotic elongation factor 1A1 (eEF1A1) as a novel substrate of TβR-I. We show that TβR-I phosphorylates eEF1A1 at Ser300 in vitro and in vivo. Ser300 was found to be important for aminoacyl-tRNA (aa-tRNA) binding to eEF1A1. Ser300 phosphorylation or mutations of Ser300 correlate with inhibition of protein synthesis in vitro and in vivo. We show that mimicking eEF1A1 phosphorylation at Ser300 results in inhibition of cell proliferation, and that mutations of Ser300 affect TGF-β dependency in inhibition of protein synthesis and cell proliferation. Increased expression of eEF1A has been reported to enhance carcinogenesis. An analysis of human breast cancer cases revealed a decrease of eEF1A1 phosphorylation at Ser300 in malignant tumor cells as compared to epithelial cells in noncancerous tissues.'] Does phosphorylation of eEF1A1 at Ser300 by TβR-I result in inhibition of mRNA translation?