Cytosolic acyl-CoA-binding proteins (ACBPs) are small proteins (ca. 10 kDa) that bind long-chain acyl-CoAs and are involved in the storage and intracellular transport of acyl-CoAs. Previously, we have characterized an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP, designated ACBP1, demonstrating the existence of a new form of ACBP in plants (M.-L. Chye, Plant Mol. Biol. 38 (1998) 827-838). ACBP1 likely participates in intermembrane lipid transport from the ER to the plasma membrane, where it could maintain a membrane-associated acyl pool (Chye et al., Plant J. 18 (1999) 205-214). Here we report the isolation of cDNAs encoding ACBP2 (Mr 38,479) that shows conservation in the acyl-CoA-binding domain to previously reported ACBPs, and contains ankyrin repeats at its carboxy terminus. These repeats, which likely mediate protein-protein interactions, could constitute a potential docking site in ACBP2 for an enzyme that uses acyl-CoAs as substrate, in vitro binding assays on recombinant (His)6-ACBP2 expressed in Escherichia coli show that it binds 14[C]palmitoyl-CoA preferentially to 14[C]oleoyl-CoA. Analysis of the acyl-CoA-binding domain in ACBP2 was carried out by in vitro mutagenesis. Mutant forms of recombinant (His)6-ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA-binding domain were less effective in binding 14[C]palmitoyl-CoA. Northern blot analysis showed that the 1.6 kb ACBP2 mRNA, like that of ACBP1, is expressed in all plant organs. Analysis of the ACBP2 promoter revealed that, like the ACBP1 promoter, it lacks a TATA box suggesting the possibility of a housekeeping function for ACBP2 in plant lipid metabolism.