Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion derivatives of ACBPI or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting. The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy whereby ACBPI was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.