Conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the toxic reactions of subsequent XO-derived superoxide, hydrogen peroxide and hydroxyl radical, have been suggested to be critical factors in several mechanisms of tissue pathophysiology. In the lung, intracellular XO-derived products may modulate type II pneumocyte surfactant turnover and barrier function, jeopardizing the pulmonary air-blood barrier. We characterized total cellular XDH/XO enzymatic activity in freshly isolated and cultured rat pulmonary type II epithelial cells. Type II cells were isolated and cultured on fibronectin-pretreated dishes, with a plating efficiency after 36 h in culture of 40% or 14% when quantified via cellular protein or DNA, respectively. Over the subsequent 96 h in culture, monolayer DNA was unchanged, whereas protein per cell increased continuously. Alterations in different cellular enzymatic activities were also detected in these cultured cells. In culture, total cellular XDH/XO and catalase activities decreased in a logarithmical fashion with respect to time, whether normalized for cellular protein or DNA. The rate of loss of these enzymes was greatest when normalized for cell protein, but was also significant when the activities were normalized for DNA. When compared to freshly isolated type II cells, catalase and total XDH/XO activities normalized for protein decreased 78% and 72%, respectively, during the first 36 h of culture. After 132 h in culture, XDH/XO and catalase activities normalized for protein decreased 93% and 84%, respectively, when compared to freshly isolated cell values. Total cellular XDH/XO activity in the oxidase form (% XO) was initially 31% in freshly isolated type II cells and increased to 67% during the 132 h culture period. In contrast to the loss of total cellular XDH/XO and catalase, no significant change in lactate dehydrogenase (LDH) activity occurred during culture of the type II cells. In type II cells the conversion of XDH to XO, the cytotoxic potential of XO, and the activity of the hydrogen peroxide scavenger, catalase, is expected to be strongly influenced by in vitro culture. Thus, strong consideration should be made before transposing information obtained from cultured type II cells to in vivo situations.