Functioning of the β-adrenergic response system is important for successful transition of the neonate from fetal life to breathing air. We characterized the β-adrenergic receptors on human fetal lung type II cells, the cell type responsible for many pulmonary responses sensitive to β-adrenergic stimulation. Type II cells were isolated from human fetal lung explants, and membrane particulates prepared from these cells were used for radioligand binding studies.125I-iodocyanopindoloI, a specific β-adrenergic antagonist, bound to a single class of saturable, high-affinity binding sites on type II cell membranes with a receptor concentration of 78 ± 9 fmol receptor/mg membrane protein, a kdof 79 ± 18 nM, and 958 ±120 receptors per cell. Binding was stereoselective withl-propranolol binding with higher affinity than the inactived-isomer. The binding site had the characteristics of a β2-adrenergic receptor. The order of potency of β-adrenergic agonists was isoproterenol > epinephrine >> norepinephrine. The β2-selective antagonist ICI 118 551 competed for a single class of high-affinity sites. Agonist binding affinity was reduced in the presence of guanyl nucleotides, consistent with receptors coupled to guanine nucleotide binding proteins. β-Adrenergic agonists also stimulated adenylyl cyclase in these membrane preparations.125I-iodocyanopindolol binding to membranes prepared from human fetal lung fibroblasts indicated fewer receptors (404 ± 68) than were present on type II cells. Work by others has suggested a difference in lung function and lung β-adrenergic receptor concentration between males and females. After culturing midgestation human fetal lung for 5 d we found no significant difference between the β-adrenergic receptor number per type II cell for male (820 ± 165) and female (1107 ± 220) specimens. (Pediatr Res 32: 350–355, 1992)