The goal was to investigate whether the diverse glucosinolate (Gl) profiles described for different Arabidopsis thaliana (L.) Heynh. ecotypes are at least partially shaped by the kinetic properties of sulfotransferases (SOTs) (EC 2.8.2.-) catalyzing the final step in Gl core structure biosynthesis. This study focuses on only one of the three SOTs that contribute to Gl biosynthesis. Homologues of AtSOT18 proteins were characterized, which was inspired by earlier findings on SOTs from ecotypes Col-0 and C24 differing in two amino acids (aa) and specific enzyme activities. Could there be a correlation of AtSOT18 enzyme activities and differences in Gl profiles between the ecotypes? SOT18 sequences from eight Arabidopsis ecotypes with highly diverse Gl patterns differed in two aa at various positions in the protein sequence. The SOT18 sequence from Col-0 showed the highest similarity to the largest number of other sequences in the alignment. The small differences in the primary sequence lead to important structural changes in secondary and tertiary structure that might be the key of different kinetic activities towards a broad range of substrates. All recombinant AtSOT18 proteins showed low substrate specificity with an indolic Gl, while the specificity for aliphatic substrates varied. There is no correlation in the kinetic behavior with the major ds-Gl contents or with the ratio of C(3)/C(4) ds-Gl in the respective ecotype. Therefore, is it unlikely that ds-Gl AtSOT18 proteins play a major role in shaping the Gl profile in Arabidopsis.