Antibodies directed against the major apoprotein associated with rabbit lung surfactant were used to characterize the induction and cellular localization of this protein during rabbit fetal lung development. In lung tissues from rabbits of 26 days gestational age and older, discrete epithelial type II cells were stained positively using the peroxidase antiperoxidase technique. The content of the major protein in homogenates of fetal lung tissue was analyzed using an immunoblotting technique. A protein of about 29 kDa, pI less than or equal to 5.6, was first detectable in fetal lung tissue on day 24 of gestation. The 29-36 kDa, mature form of the surfactant apoprotein was first detectable in lung homogenates from 30-day gestational age fetal rabbits. Treatment of homogenates of day 26 and 31 fetal lung tissues with endoglycosidase F, yielded, in both cases, an immunoreactive triplet with more neutral isoelectric points than the proteins in the untreated homogenates. By immunoblot analysis, we found that only the 29-36 kDa, mature form of the surfactant apoprotein was present in lamellar bodies purified from lung tissues of fetuses of 28 and 31 days and from day 2 neonates. These findings are suggestive that only the mature, 29-36 kDa form of the surfactant apoprotein is associated with lamellar bodies during fetal lung type II cell differentiation in vivo.