Amino acids 44-53 of mouse vascular smooth muscle alpha-actin are encoded by a region of exon 3 that bears structural similarity to an essential MCAT enhancer element in the 5' promoter of the gene. The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, interact with each other and with opposite strands of the enhancer to repress transcription in fibroblasts (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436; Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). In this study, we employed both recombinant and fibroblast-derived proteins to demonstrate that all three proteins specifically interact with the mRNA counterpart of the exon 3 sequence in cell-free binding assays. When placed in the 5'-untranslated region of a reporter mRNA, the exon 3-derived sequence suppressed mRNA translation in transfected fibroblasts. Translational efficiency was restored by mutations that impaired mRNA binding of Puralpha, Purbeta, and MSY1, implying that these proteins can also participate in messenger ribonucleoprotein formation in living cells. Additionally, primary structure determinants required for interaction of Purbeta with single-stranded DNA, mRNA, and protein ligands were mapped by deletion mutagenesis. These experiments reveal highly specific protein-mRNA interactions that are potentially important in regulating expression of the vascular smooth muscle alpha-actin gene in fibroblasts.