AbstractAnalysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys‐98 with Cys‐125, and Cys‐166 with Cys‐200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18–33% residue identities) from microorganisms. However, one half‐Cys (i.e., Cys‐166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N‐terminal part of the tomato pectinesterase, another in the C‐terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to highlight a functional site in a midchain segment.