Cell−cell interactions are important in embryogenesis, in adult physiology and pathophysiology of many disease processes. Co-cultivation of parenchymal and mesenchymal cells has been widely utilized as a paradigm for the study of cell−cell interactions in vitro. In addition, co-cultures of two cell types provide highly functional tissue constructs for use in therapeutic or investigational applications. The inherent complexity of such co-cultures creates difficulty in characterization of cell−cell interactions and their effects on function. In the present study, we utilize conventional “randomly distributed” co-cultures of primary rat hepatocytes and murine 3T3-J2 fibroblasts to investigate the role of increasing fibroblast density on hepatic function. In addition, we utilize microfabrication techniques to localize both cell populations in patterned configurations on rigid substrates. This technique allowed the isolation of fibroblast number as an independent variable in hepatic function. Notably, homotypic hepatocyte interactions were held constant by utilization of similar hepatocyte patterns in all conditions, and the heterotypic interface (region of contact between cell populations) was also held constant. Co-cultures were probed for synthetic and metabolic markers of liver-specific function. The data suggest that fibroblast number plays a role in modulation of hepatocellular response through homotypic fibroblast interactions. The response to changes in fibroblast number are distinct from those attributed to increased contact between hepatocytes and fibroblasts. This approach will allow further elucidation of the complex interplay between two cell types as they form a functional model tissue in vitro or as they interact in vivo to form a functional organ.