Rationale: Esophageal manometry is the clinically available method to estimate pleural pressure, thus enabling calculation of transpulmonary pressure (PL). However, many concerns make it uncertain in which lung region esophageal manometry reflects local PL. Objectives: To determine the accuracy of esophageal pressure (Pes) and in which regions esophageal manometry reflects pleural pressure (Ppl) and PL; to assess whether lung stress in nondependent regions can be estimated at end-inspiration from PL. Methods: In lung-injured pigs (n = 6) and human cadavers (n = 3), Pes was measured across a range of positive end-expiratory pressure, together with directly measured Ppl in nondependent and dependent pleural regions. All measurements were obtained with minimal nonstressed volumes in the pleural sensors and esophageal balloons. Expiratory and inspiratory PL was calculated by subtracting local Ppl or Pes from airway pressure; inspiratory PL was also estimated by subtracting Ppl (calculated from chest wall and respiratory system elastance) from the airway plateau pressure. Measurements and Main Results: In pigs and human cadavers, expiratory and inspiratory PL using Pes closely reflected values in dependent to middle lung (adjacent to the esophagus). Inspiratory PL estimated from elastance ratio reflected the directly measured nondependent values. Conclusions: These data support the use of esophageal manometry in acute respiratory distress syndrome. Assuming correct calibration, expiratory PL derived from Pes reflects PL in dependent to middle lung, where atelectasis usually predominates; inspiratory PL estimated from elastance ratio may indicate the highest level of lung stress in nondependent baby lung, where it is vulnerable to ventilator-induced lung injury.