While all the alkylation methods commonly used in the chemistry of classical calixarenes failed, tetranitrothiacalix[4]arene was easily alkylated using various alcohols under Mitsunobu reaction conditions. The products thus obtained were immobilized in the 1,3-alternate conformation as suggested by ¹H NMR and proven unequivocally by X-ray analysis. The introduction of chiral alkyl substituents into the lower rim of thiacalixarene gave us an opportunity to form well-preorganized ureido cavities on both sites of the system. As revealed by ¹H NMR titration experiments, such compounds were capable of chiral anion recognition even in DMSO-d₆ which is a highly competitive solvent towards hydrogen bonding interactions. The highest chiral discrimination was achieved for free serine with a selectivity factor of 3.13 for the D-isomer.