The stimulation of cellular cholesterol and phospholipid efflux by apolipoprotein A-I is mediated by the activity of the ATP-binding cassette transporter A1 (ABCA1). Individuals with Tangier disease harbor loss-of-function mutations in this transporter that have proven useful in illuminating its activity. Here, we analyze a mutation that deletes the last 46 residues of the 2261 amino acid transporter (Delta46) and eliminates its lipid efflux. As the final four amino acids of the C terminus represent a putative PDZ-binding motif, we initially characterized deletion mutants lacking only these residues. Although a moderate decline in lipid efflux was detected, this decline was not as profound as that seen in the Delta46 mutant. Subsequent systematic analysis of the ABCA1 C terminus revealed a novel, highly conserved motif (VFVNFA) that was required for lipid efflux. Alteration of this motif, which is present in some but not all members of the ABCA family, did not prevent trafficking of the transporter to the plasma membrane but did eliminate its binding of apoA-I. Chimeric transporters, generated by substituting the C termini of either ABCA4 or ABCA7 for the endogenous terminus, demonstrated that ABCA1 could stimulate cholesterol efflux without its PDZ-binding motif but not without the VFVNFA motif. When a peptide containing the VFVNFA sequence was introduced into ABCA1-expressing cells, ABCA1-mediated lipid efflux was also markedly inhibited. These results indicate that the C-terminal VFVNFA motif of ABCA1 is essential for its lipid efflux activity. The data also suggest that this motif participates in novel protein-protein interactions that may be shared among members of the ABCA family.