In vertebrates, there are two variants of eukaryotic peptide elongation factor 1A (eEF1A; formerly eEF-1 ), eEF1A1 and eEF1A2, which have three well-conserved domains (DI, DII, and DIII). In neurons, eEF1A1 is the embryonic type, which is expressed during embryonic development as well as the first two postnatal weeks. In the present study, EGFP-tagged eEF1A1 truncates were expressed in cortical neurons isolated from rat embryo (E18-19). Live cell images of transfected neurons showed that DIII-containing EGFP-fusion proteins (EGFP-DIII, -DII-III, -DI-III) formed clusters that were confined within somatodendritic domains, while DIII-missing ones (EGFP-DI, -DII, -DI-II) and control EGFP were homogeneously dispersed throughout the neuron including axons. In dendrites, EGFP-DIII was targeted to the heads of spine- and filopodia-like protrusions, where it was colocalized with SynGAP , a postsynaptic marker. Our data indicate that DIII of eEF1A1 mediates formation of clusters and localization to spines. [BMB reports 2012; 45(4): 227-232]