Culturing cells in 3-dimensional (3D) systems is important in tissue engineering and in fundamental studies of cellular mechanisms that are sensitive or specific to the 3D environment. To guide the engineering of artificial vocal fold lamina propria tissue, we developed 3D cultures containing human vocal fold fibroblasts (hVFFs) dispersed in a synthetic peptide hydrogel matrix. Growth factors were added to the culture to examine their influence on extracellular matrix (ECM) synthesis, cell proliferation, and matrix contraction. The hVFF-hydrogel constructs were treated with transforming growth factor-beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF), or hepatocyte growth factor (HGF), and the culture was maintained for 21 days. TGF-beta1 induced matrix contraction and enhanced collagen and sulfated glycosaminoglycan production, bFGF effectively increased cell proliferation, and HGF stimulated synthesis of hyaluronic acid and elastin with less collagen accumulation than other conditions. Of the growth factors tested, HGF appears to be most useful for stimulating essential tissue components for restoring vocal fold pliability. The results also suggest that multiple growth factors might be employed sequentially or in combination to program the makeup of cell-hydrogel constructs for vocal fold tissue repair.