Fibronectin (FN) production was quantified in the extracellular medium and in extracts of human diploid fibroblasts (HDF) by enzyme-linked immunosorbent assay and by immunoprecipitation of [35S]methionine biosynthetically labeled proteins. FN output was increased in normal late passage (old) HDF and in prematurely senescent Werner syndrome (WS) fibroblasts, compared to normal early passage (young) HDF. Output was maximal when cells were preconfluent, exceeding the level found at confluence and postconfluence. For all cell types the highest proportion of newly synthesized FN was found in the extracellular compartment. Both young and old HDF exposed to regular growth medium containing 15% fetal bovine serum (FBS) secreted twice as much FN as when exposed to serum-free medium, and cognate mRNA levels were commensurate with protein output. WS fibroblasts secreted 1.6-fold more FN than old HDF when both cell types were exposed to medium containing 15% FBS. Immunofluorescent analysis revealed greater association of FN with WS and old HDF than with young HDF. Furthermore, WS cells exposed to 15% FBS contained 3.4-fold more FN mRNA and produced seven times more FN protein compared to WS cells exposed to serum-free medium. Thus, WS fibroblasts secrete more FN than old normal and young normal HDF in the presence of 15% FBS due to augmentation of FN mRNA levels and enhanced efficiency of FN mRNA translation and/or post-translational processing.