Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana, three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.