Lung tissue from fetal rabbits at 24 days' gestation was maintained in organ culture in defined medium. This system has been used to study the effect of dexamethasone on general protein synthesis and on the synthesis of specific proteins by fetal rabbit lung in vitro. Glucocorticoid treatment had no effect on the overall incorporation of labeled amino acids into protein. However, it increased the incorporation of [35S] methionine into 5 of the nearly 400 proteins catalogued by two-dimensional gel electrophoresis. When tissue was taken from fetuses at 20 and 22 days' gestation, it was found that the effect of dexamethasone on the incorporation of [35S] methionine into some of these five peptides was dependent on the gestational age of the animals at the time of sacrifice. When dexamethasone was administered for 2 consecutive days to pregnant rabbits beginning on gestational day 25, two-dimensional gels of [35S] methionine-labeled slices of fetal lungs revealed several glucocorticoid treatment in vitro. Following isolation of type II cells from fetal lungs after glucocorticoid administration in vivo and labeling the cells with [35S] methionine, two-dimensional gels of cell lysates exhibited several changes, some of which are identical to the changes seen in whole lung after glucocorticoid treatment in vivo or in vitro and some that appear to be unique to type II cells.