Abstract In mammals, long-chain acyl-CoA synthetases (ACSL) are necessary for fatty acid degradation, phospholipid remodeling, and production of long acyl-CoA esters that regulate various physiological processes. These enzymes play a crucial role in plasma membrane phospholipid turnover in erythrocytes, maintaining the complex phospholipid molecular species composition essential for proper membrane function. The mechanism by which this highly dynamic turnover together with an ever-changing plasma fatty acid pool maintains phospholipid composition is poorly understood. We have previously cloned Acyl-CoA Synthetase Long-chain member 6 (ACSL6), the isoform responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants of this protein were subsequently isolated from brain and testis. We report the expression of four different variants of ACLS6 in reticulocytes, one as we originally reported, two of which are novel, one as was identified in brain cells. PCR amplifications using primers for the predicted variable regions were performed from cDNAs of CD34 positive erythroid progenitors, K562 cells, fetal blood cells, reticulocytes and placenta. ACSL variants were expressed in E. coli host BL21DE3 cells using the pET28a vector, and detected by His tag immuno detection. Sequence alignments were generated using sequences retrieved from RefSeq and GenBank databases on the NCBI site. Exon and intron definition for ACSL members were obtained using evidence viewer and model maker available at the map viewer page of each gene. We identified four different spliced variants of ACSL6 in erythroid cells based on a mutually exclusive exon pair. Each exon of this pair encodes a slightly different short motif that contains the fatty acid Gate domain, a conserved structural domain found in all vertebrate and invertebrate ACSL homologs. The motif differs in the presence of either the aromatic residue phenylalanine (Phe) or tyrosine (Tyr), and seems to play a role in substrate specificity. One of the new forms contained an exon not found in any other ACSL isoforms. Erythroid precursors also express the closely related ACSL1, and we characterized two additional isoforms of this protein, similar to ACSL6. When analyzed on denaturing SDS polyacrylamide gel both ACSL1 and 6 appeared to exist as a dimer. Based on our results, we propose the generation of two different Gate-domains by alternative splicing of the two exons in these proteins. One represents a switch of the Phe to the Tyr Gate-domain motif, the other resulted of the exclusion of both. Swapping of this motif appears to be common to all mammalian homologs of ACSL1 and 6. We conclude that the Phe to a Tyr substitution in the Gate-domain, or its removal, together with the formation of homo or heterodimers will allow ACSL6 the structural diversity to define substrate specificity that maintains the complex plasma membrane phospholipid molecular species composition in erythrocytes.