AbstractThe purpose of this investigation was to examine the suitability of an organotypic lung‐cell culture model for the study of factors influencing fetal lung‐cell differentiation. It has been reported that the use of carbonstripped (hormone‐depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiationin vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon‐stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three‐dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbonstripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that (1) the hormones extracted from bovine fetal calf serum by carbonstripping are not solely responsible for the continued fetal lung cell differentiation observedin vitro, and (2) that spatial relationships between lung cellsin vitromay be a significant factor in the control of differ