Earlier studies of the fetal lung in tissue culture have led to the conclusion that its maturation is autonomous. We have studied the role of the small amount of cortisol supplied by the serum routinely added to culture media. It was observed that 28-day-old fetal rabbit lung cells continued to grow and differentiate in culture medium supplemented with 10% calf serum (2.2 X 10(-8) M cortisol). However, when these cells were propagated in medium supplemented with calf serum which was charcoal stripped or from adrenalectomized animals, growth and differentiation were prevented (<1 X 10(-12) M cortisol). The addition of cortixol (1 X 10(-10) to 1 X 10(-5) M) to such media restored the ability of these cells to grow and differentiate in culture. Other classes of steroid hormones failed to restore such activity even with a 10-fold excess of cortisol. We conclude that maturation of the 28-day-old fetal rabbit lung in vitro is dependent upon the presence of cortisol.