Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis-acting sequences in the 3' untranslated regions (3'UTRs) of the mRNAs concerned acting together with trans-acting proteins. The C-MYC transcription factor is a proto-oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c-myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3'UTR. Here we show the specific binding of a trans-acting factor to the perinuclear localization element in the 3'UTR of c-myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross-linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c-myc 3'UTR implicated in localization; a protein of approximately 36 kDa exhibited specific, Ca(2+)-dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA-affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA-protein complex formed by cell extracts was further retarded by anti-(annexin A2). Purified annexin A2 bound to the same region of the c-myc 3'UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA-binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.