The adenosine-uridine (AU)-rich sequences within the 3' untranslated region (UTR) of many short-lived mRNAs are important in their rapid degradation. We present evidence that human embryonic lung fibroblasts (W138) contain five major proteins of 70, 45, 40, 38, 32.5 kd, which specifically bind the AU-rich region of human granulocyte-macrophage colony-stimulating factor (GM-CSF) 3'UTR containing 7 x AUUUA motifs. The 40 and 38 kd proteins also bound the 3x and 5 x AUUUA cassettes and even more strongly bound to the AUUUUUUUA motif. All five of these proteins showed more abundant localization in the nucleus than the cytoplasm. The 32.5 kd protein was the major cytoplasmic AU-binding protein. Incubation with actinomycin D resulted in a marked increase in binding activity of 45, 40, 38, and 32.5 kd proteins in the cytoplasm, accompanied by decreased binding activity of the 32.5 kd protein in the nucleus. Antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) immunoprecipitated the 40 and 38 kd proteins, and antibody against the AU-rich element RNA-binding protein (AUF1) immunoprecipitated the 45, 40, and 38 kd proteins. The present results not only demonstrated that hnRNP C are AU-binding proteins which are present in the cytoplasm as well as the nucleus, but another group of AU-binding proteins (AUF1 [45, 40, 38 kd], and 32.5 kd), which are not hnRNP, have characteristics related to those of hnRNPs. Taken together with our previous results (Akashi et al., 1994, Blood, 83:3182-3187), AU-binding factors including hnRNP C and AUF1, which bind more than 3 x AUUUA motifs, may be involved in rapid degradation of these transcripts. No significant quantitative changes of these proteins in their binding activity to AU-rich sequences occurred in response to several stimuli that stabilize GM-CSF mRNA, indicating that binding of these proteins to their cognate RNA is not responsible for the stabilization of these transcripts.