Recent studies have established that cultured human skin fibroblasts secrete the soluble precursor of elastin, tropoelastin (TE). The present studies evaluate, by an enzyme-linked immunosorbent assay, the stability of the TE phenotype and the effect of culture conditions and donor age on TE accumulation by human skin fibroblasts. Tropoelastin was maximally produced by 2 control fibroblast strains at early confluency (32-49 X 10(3) molecules/cell/h), and its serum-dependent accumulation in the medium was linear for at least 72 h. Inhibition of cross-linking had no effect on the rate of elastin production. Optimum serum concentrations for TE production differed for fibroblast cell strains derived from foreskin and trunk skin fibroblasts. Production of TE by human skin fibroblasts was stable through nearly 30 population doublings after which there was a greater than 2-fold decline in the rate of accumulation. In a cohort of donor strains, TE production appeared to decline at donor ages greater than or equal to 70 years. Under standard culture conditions, cell strains from normal donors of various ages produced TE at rates ranging from 25-69 X 10(3) molecules/cell/h. Rates of TE accumulation in medium were not significantly altered by degradation of TE, as a variety of cell strains tested exhibited minimal cell-associated elastolytic activity. Based on the demonstration of a stable elastin phenotype, skin fibroblast cultures provide a new system for studying regulation of elastin biosynthesis and evaluating potential defects in elastin metabolism associated with certain connective tissue disorders.