Pulmonary carbonic anhydrase (CA) activity plays important roles in carbon dioxide exchange, fluid secretion, and pH regulation. This study reports the use of molecular and immunologic techniques to characterize expression of the high-activity cytosolic isoenzyme CA II in rat lung tissue. Northern blot analysis of RNA isolated from various rat tissues revealed that the lung is a site of abundant tissue-specific CA II gene expression. The cell type primarily responsible for CA II expression in the lung was identified by immunohistochemistry as the alveolar type II pneumocyte. RNA blot and immunoblot analyses of isolated rat type II cells in culture confirmed CA II expression by this cell type. Little immunoreactive CA I and no CA IV was detected in these cells. Inhibition studies confirmed that the majority of CA activity in isolated type II cells is attributable to CA II. CA II expression was found to continue in these cells beyond 72 h in culture, a timeframe during which these cells had dedifferentiated. The ontologic pattern of CA II expression in the lung was found by RNA blot analysis to be disparate from that of the surfactant-associated proteins. These observations suggest roles for CA II in alveolar pneumocytes independent of (or in addition to) participation in surfactant biology. Such roles may include the regulation of fluid secretion or facilitation of carbon dioxide elimination.