The aim of this study was to assess the association between regional tidal volume (Vt), regional functional residual capacity (FRC), and the expression of genes linked with ventilator-induced lung injury. Two groups of BALB/c mice (n = 8 per group) were ventilated for 2 hours using a protective or injurious ventilation strategy, with free-breathing mice used as control animals. Regional Vt and FRC of the ventilated mice was determined by analysis of high-resolution four-dimensional computed tomographic images taken at baseline and after 2 hours of ventilation and corrected for the volume of the region (i.e., specific [s]Vt and specific [s]FRC). RNA concentrations of 21 genes in 10 different lung regions were quantified using a quantitative PCR array. sFRC at baseline varied regionally, independent of ventilation strategy, whereas sVt varied regionally depending on ventilation strategy. The expression of IL-6 (P = 0.04), Ccl2 (P < 0.01), and Ang-2 (P < 0.05) was associated with sVt but not sFRC. The expression of seven other genes varied regionally (IL-1β and RAGE [receptor for advanced glycation end products]) or depended on ventilation strategy (Nfe2l2 [nuclear factor erythroid-derived 2 factor 2], c-fos, and Wnt1) or both (TNF-α and Cxcl2), but it was not associated with regional sFRC or sVt. These observations suggest that regional inflammatory responses to mechanical ventilation are driven primarily by tidal stretch.