Ozone is a highly reactive gas present in urban air, which penetrates deep into the lung and causes lung injury. The alveolar epithelial cells are among the first cell barriers encountered by ozone. To define the molecular basis of the cellular response to ozone, primary cultures of rat alveolar type II and type I-like cells were exposed to 100 ppb ozone or air for 1 h. The mRNA from both phenotypes was collected at 4 and 24 h after exposure for gene expression profiling. Ozone produced extensive alterations in gene expression involved in stress and inflammatory responses, transcription factors, antioxidant defenses, extracellular matrix, fluid transport, and enzymes of lipid metabolism and cell differentiation. Real-time reverse transcription–polymerase chain reaction and Western blot analysis verified changes in mRNA and protein levels of selected genes. Besides the increased stress response, ozone exposure downregulated genes of cellular differentiation. The changes were more prominent at 4 h in the type I-like phenotype and at 24 h in the type II phenotype. The type I-like cells were more sensitive to ozone than type II cells. The genome-wide changes observed provide insight into signal pathways activated by ozone and how cellular protection mechanisms are initiated.