Composition and architecture of the extracellular matrix dictate cell behavior. Proteoglycans bind multiple components of the extracellular matrix by serving as important regulators of cell behavior. Given the influence of culture architecture on cell function, we investigated whether switching NIH3T3 fibroblasts from growth on type 1 collagen in monolayer to a collagen gel might influence dermatan sulfate expression. Immunofluorescent staining, immunoblot, and Western blot demonstrated an induction in decorin expression in cells switched to collagen gels. This induction was associated with a 40-fold increase in decorin transcript expression determined by quantitative real time PCR. Disaccharide analysis of extracted glycosaminoglycans from collagen gels showed an increase in total glycosaminoglycan and in the ratio of chondroitin sulfate to heparan sulfate compared with monolayer culture. The ratio of chondroitin sulfate to heparan sulfate likewise increased on syndecan-1 from gel culture. Digestion with chondroitinase B showed that this induced chondroitin sulfate was dermatan sulfate. Syndecan-1 extracted from wounded mouse skin also displayed an increase in dermatan sulfate synthesis compared with unwounded skin. Furthermore, glycosaminoglycans from collagen gel culture activated keratinocyte growth factor, whereas glycosaminoglycans from monolayer culture lacked this ability. These findings suggest that regulation of dermatan sulfate and dermatan sulfate proteoglycan is dependent on extracellular matrix architecture. The ability of collagen gel culture to mimic better the in vivo dermal environment may be due in part to this influence on dermatan sulfate and dermatan sulfate proteoglycan synthesis.