Abstract Surface analysis and chromatographic data are used to examine the structure of surface groups in a reversed-phase chromatographic environment involving an octadecyl-derivatized silica substrate. The support is characterized by 29Si solid-state nuclear magnetic resonance spectroscopy and surface titration, and results are compared to other spectroscopic methods for the quantitation of surface species. Quantitative nuclear magnetic resonance data permits examination of the number and reactivity of single versus geminal hydroxyl sites as a function of octadecyldimethylsilyl coverage. It is postulated that an acidic, reactive subset of surface silanols exists that includes a large proportion of the geminal silanol sites, and a subset of the single silanol sites including hydrogen-bonded species such as the vicinal silanols. Knowledge of surface structure helps to explain various types of chromatographic behavior including the inaccessibility of some surface region silanols at moderate silane coverages, the partition-like behavior of octadecyl substrates, and the pH-dependent retention of non-polar solutes in reversed-phase chromatography.