Posttranscriptional controls play a major role in eucaryotic gene expression. These controls are mediated by sequence-specific interactions of cis-acting determinants in target mRNAs with one or more protein factors. The positioning of a subset of these mRNA-protein (RNP) complexes within the 3′ untranslated region (3′ UTR) may allow them to remain associated with the mRNA during active translation. Robust expression of human α-globin mRNA during erythroid differentiation has been linked to formation of a binary complex between a KH-domain protein, αCP, and a 3′ UTR C-rich motif. Detection of this “α-complex” has been limited to in vitro studies, and the functional state of the α-globin mRNA targeted by αCP has not been defined. In the present study we demonstrate that a significant fraction of αCP is associated with polysomal mRNA. Targeted analysis of the polysomal RNP complexes revealed that αCP is specifically bound to actively translating α-globin mRNA. The bound αCP is restricted to the poly (C)-rich 3′ UTR motif and is dislodged when ribosomes are allowed to enter this region. These data validate the general importance of the 3′ UTR as a sheltered site for RNP complexes and support a specific model in which the stabilizing function of αCP is mediated on actively translating target mRNAs.