The overall objective of this investigation was to characterize the extracellular matrix deposited by the stromal fibroblasts as a function of time in culture and matrix microstructure. Stromal fibroblasts were seeded onto collagen matrices and cultured for up to 5 weeks. The collagen matrices contained collagen fibrils with an average diameter of 215±20 nm. When cultured on a collagen film, an average fibril diameter of 62±39 nm was observed for single layer films with only slight variations with time in culture, and after 1 week of culture between two film layers 67±47 nm fibrils were observed after 1 week. When the film surface was molded into 1 and 2 lm microgrooves, the initial average fibril diameter of the extracellular matrix was 73±21 and 73±31 nm respectively. When cultured on a collagen sponge, an average fibril diameter of 107±20 nm was initially observed and decreased to 47.5±17 nm after 1 week in culture. For cells cultured on a collagen sponge, Western blotting showed an increase in myofibroblast phenotype expression with time in culture. Shifts in phenotype were less distinct for cells cultured on collagen films. The microstructure, rather than geometry, of the matrix substrate appeared to influence the newly synthesized extracellular matrix and cell phenotype.