Previously, we have shown that the vimentin 3′ untranslated region (3′ UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as 'bait' to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA-protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1c and hRIP, code for proteins of a size consistent with in vitro cross-linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-Iγ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin's 3′ UTR. Both in vivo synthesized eEF-1γ and HAX-1 proteins were 'pulled out' of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin's 3′ UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.