We have examined thein vivotargeting potential of the Pleckstrin Homology (PH) domain from human βIΣII spectrin using a novelAequoria victoriagreen fluorescent protein (GFP) fusion vector constructed from a human codon optimized cDNA. This vector efficiently expresses both GFP and the GFP spectrin fusion protein in COS7 and other cell lines. GFP expressed alone shows only diffuse cytoplasmic staining which is not associated with the plasma membrane. In contrast the GFP-βIΣII spectrin PH domain fusion protein localizes under the plasma membrane of transfected COS7 cellsin vivo.Fixation of cells transfected with GFP alone in −20°C methanol results in the removal of all specific fluorescence. In contrast cells transfected with the GFP-βIΣII spectrin construct and fixed −20°C methanol continue to show strong membrane fluorescence, consistent with a role for the spectrin PH domain in membrane localizationin vivo.