Glyoxysomal 3-ketoacyl-CoA thiolase is the last enzyme in the β-oxidation of fatty acids in plant glyoxysomes. A full-length cDNA of the glyoxysomal 3-ketoacyl-CoA thiolase from Brassica napus and a truncated version, lacking the N-terminal targeting signal were cloned in a T7 promoter-based vector. Both recombinant proteins were expressed in Escherichia coli and activity was measured. Full-length and truncated 3-ketoacyl-CoA thiolase have comparable activity in E. coli. Moreover, full-length 3-ketoacyl-CoA thiolase was purified from E. coli and N-terminal sequencing of the protein confirmed that the precursor form indeed is enzymatically active.