Long-chain acyl-CoA synthetases (LACSs) activate fatty acids for further metabolism and are encoded by a multi-gene family in Arabidopsis. AtLACS6 possesses a type 2 (PTS2) peroxisomal targeting sequence, whilst AtLACS7 has both a type 1 and type 2 peroxisomal targeting sequence. AtLACS7 was used as bait in a yeast two-hybrid screen. Multiple clones of the PTS1 receptor PEX5 were isolated. Quantitative beta-galactosidase assay indicated that full-length PEX5 interacts with AtLACS7 with higher affinity than the TPR domains alone. The interaction between PEX5 and AtLACS7 was confirmed by co-immunoprecipitation and shown to be specific for the PTS1, therefore the AtLACS7 PTS1 is accessible to bind PEX5 in the full-length AtLACS7 protein. The expression profile of AtLACS6, AtLACS7, AtPEX5, and AtPEX7 revealed that AtLACS6 and 7 have distinct patterns of expression and we speculate that the possession of two targeting signals may be advantageous for the import of AtLACS7 when receptors may be limiting.