Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. Mn-55 electron-nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, similar to 3.5 T) to determine the Mn-55 nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the Mn-55 ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e(2)Qq/h, are significantly different; 10.7 +/- 0.6 MHz for Mn-B(2+.) The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for Mn-A(2+) and -263.5 MHz for Mn-B(2+.) The principle z-axis for Mn-A(2+) is not aligned with any of the Mn-ligand directions, but is 25% off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of Mn-B(2+) the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90 degrees with respect to Mn-A. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e(2)Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally.