Fetal calf ligamentum nuchae fibroblasts, cultured from animals of different gestational age, synthesize a unique, low molecular weight collagen termed FCL-1 (Sage, H., Mecham, R., Johnson, C., and Bornstein, P., 1983, J. Cell Biol. 97:1933-1938). Previous studies on the elastogenic differentiation of these cells in vitro demonstrated that the extracellular matrix (ECM) protein elastin was specilically induced in undifferentiated fibroblasts when they were grown on ligament ECM isolated from animals at later stages of development (Mecham. R.P., Madaras, J.G., and Senior, R.M., 1984. J. Cell Biol. 98:1804-1812). To investigate the expression of FCL-1 as a function of developmental age, we grew fetal calf ligament fibroblasts from an 8.5 d (first trimester) animal (FCL 85d) on three different substrata: ligament from a 120 d (second trimester) animal, ligament from a 270 d (term) animal, and unmodified plastic tissue culture dishes. FCL 270d fibroblasts were grown on plastic substrata and served as a differentiated cellular control. Analysis of metabolically radiolabeled proteins from both the culture media and the cell layers showed that the synthesis of FCL-1 was selectively increased in those cells cultured on ligament ECM. For FCL 85d fibroblasts grown on 120 d and 270 d ligaments, FCL-1 comprised 17% and 22%, respectively, of the culture medium proteins that precipitated at concentrations of ammonium sulfate from 20-50%. FCL 85d and 270d fibroblasts grown on plastic substrata yielded values of 2.5% and 1.0%, respectively. This effect appeared to be specific for this collagen and did not reflect a general increase in the synthesis of connective tissue proteins of the ECM (e.g., types I and III procollagen). As percent of total newly-synthesized cellular protein, the output of FCL-1 was 10-fold higher by FCL 85d cells grown on 270d ligament ECM (5.8%) as compared to that of the same cellular population grown on a plastic surface (0.56%). The presence of the ligament ECM also altered the levels and distribution of secreted proteins between the culture medium and the cell layer. These studies provide evidence for differential expression of the novel collagen FCL-1 by FCL fibroblasts during development and suggest that such expression is affected, at least in part, by interaction of the cell with a ligament ECM.