The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human ␣-globin mRNA 3 untranslated region (␣-3UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the ␣-3UTR with a variety of cytosolic extracts. This so-called ␣-complex is sequence specific and is not formed on the 3UTR of either -globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize ␣-globin mRNA in vivo result in a parallel disruption of the ␣-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of ␣-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the ␣-complex. Three predominant proteins are isolated by ␣-3UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for ␣-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the ␣-3UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of ␣-globin mRNA.Degradation of mature mRNA in eukaryotic cells is a regulated process that can be a significant determinant of gene expression. This regulatory process is seen in fertilized oocytes (5, 56) as well as in highly differentiated tissues (13,55,60). Multiple mechanisms underlie this regulation (12,25,50). cisregulating elements can be found in the 5Ј untranslated region (5ЈUTR) (43), the coding region (41, 52), and the 3ЈUTR (28, 60) and often affect the size of the poly(A) tract (5, 39, 56). trans-acting proteins which specifically bind to certain of these cis elements have been identified (29,42,50). An emerging theme is the paramount importance of the 3ЈUTR in control of mRNA stability and subsequent determination of gene expression (28).Recent studies have emphasized a wide spectrum of functions which are mediated by the mRNA 3ЈUTR. This region can act as a trans-acting regulator of growth and differentiation in mouse myoblasts (45, 46), a localization determinant (21) and translational regulator in Drosophila embryos (22), a control element in sex determination of the germ cell precursors in Caenorhabditis elegans (3), and a translational regulator of cell cycle-related mRNAs in Xenopus laevis (56). However, the majority of studies on the 3ЈUTR focus on its role in determination of mRNA stability (15,28,56,60). The most clearly defined example of this control is the iron-responsive element loc...