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The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). While formation and utilization of acyl-CoA takes place at the membrane, Acyl-CoA Binding Domain containing proteins (ACBD) in RBC cytosol bind acyl-CoA, limiting product feedback inhibition on ACSL and distribute Acyl-CoA to the various acyl-utilizing enzymes while protecting the cells against its potent detergent character. We have identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC, a protein with several isoforms that acts as a dimer. To relate its structure to activity, we report the expression of different modified forms in E. coli. Our data indicate that, despite the observed activity, enzyme studies of these mammalian membrane proteins in the host E. coli are strongly hampered by their aggregation into inclusion bodies. While activity can be measured, data on enzyme kinetics and specificity are questionable. Oleoyl-CoA formation from oleic acid, CoA and ATP reveled that the two transmembrane spanning segments predicted at the amino-terminus of the protein are not essential for its activity. Moreover, they are not essential for dimer formation and strong association with membranes. ACSL6 appears to be an integral membrane protein. One of the five spliced isoforms of ACSL6 reported, lacks the so-called fatty acid Gate-domain, and appears to be unable to activate long chain fatty acids. We hypothesize that this form modulates activity of the other active isoforms of ACSL6 through hetero-dimer formation. An EST clone of erythroid precursor cells identified ACBD6 as a potential AcylCoA binding protein in RBC. This modular protein contains an Acyl-CoA binding domain at the amino-terminus and two Ankyrin-repeat motifs (ANK) at the C-terminus. Both the full-length protein and the N-terminus domain were soluble when expressed and purified in E. coli. Expression of the C-terminus domain by itself rendered an insoluble protein. We report that ACBD6 binds long-chain acyl-CoA with a preference for C18:1-CoA over C20:4 and C16:0-CoA and does not bind fatty acid. Truncation of the ANK domain had no effect on the binding activity of the N-terminus domain. Together our findings implicate ACBD6 as part of the Acyl-CoA turnover mechanism in RBC, its actual role on the kinetics of ACSL and/or LPLAT activity needs to be established. The description of these proteins involved in Acyl-CoA turnover in RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.