Pulmonary interstitial fibroblasts were isolated from 4-day neonatal rat lung and cultured for varying periods of time after first passage. The ability of the cells to synthesize both soluble and insoluble elastin was examined by biochemical, immunological, and ultrastructural techniques. The cells synthesize a tropoelastin of 75,000 daltons which is cross-linked in the extracellular matrix to form sufficient amounts of insoluble elastin to be characterized directly by amino acid analysis. Although the cells produce a soluble form of elastin immediately after confluency and the deposition in the matrix begins shortly thereafter, the actual chemical definition of alkali-resistant elastin does not occur until 4 weeks after confluency. The apparent molecular weight of tropoelastin synthesized by the cells agrees with the molecular size of tropoelastin found in organ culture of neonatal lungs. The pulmonary fibroblast cultures provide an important model system for examining the regulation and response of elastin synthesis in lung interstitium.