There is increasing evidence to suggest that fetal lung growth requires normal fetal breathing movements. To study this process in vitro, the effect of mechanical stretch on proliferation of fetal rat lung cells maintained in organotypic cultures was examined. In previous studies it has been demonstrated that DNA synthesis and cell division are stimulated by stretch. To determine whether stretched cells release soluble growth factors into their culture medium, conditioned media (CMs) were collected after static culture or culture while stretched. At a 10% dilution (v/v), CM from stretched cells (S-CM) increased [3H]thymidine incorporation into DNA of mixed fetal rat lung cells by 60%, compared to CM from nonstretched cells (C-CM) (p < .01). S-CM increased [3H]thymidine incorporation of fetal lung epithelial cells by 3 to 4-fold (p < .01) and increased cell number by 11.9% (p < .05), but had no effect on fetal lung fibroblast growth. Addition of either PDGF-BB (20 ng/mL), IGF-I (25 ng/mL), or EGF (50 ng/mL) to C-CM, did not mimic the effect of S-CM on epithelial cell DNA synthesis. The stimulatory activity of S-CM on epithelial cell proliferation was heat-, acid- and trypsin-sensitive. It is concluded that organotypic fetal lung cell cultures respond to stretch by elaborating growth-promoting factors which stimulate fetal rat lung epithelial cell, but not fibroblast, proliferation.