Extracellular matrix is indispensable for cell differentiation in vivo. The aim of this study is to examine effects of extracellular matrix on adrenocortical cells embedded in collagen gel in terms of cellular proliferation and differentiation. Dissociated human or bovine adrenocortical cells were embedded in collagen gel; the gel was then immediately thin-coated on the bottom of culture dishes. The cortical cells were thus trapped in collagen and cultured in monolayer, a suitable means for observation of culture cells. To the authors' best knowledge, this "collagen gel-embedded" monolayer culture is described here for the first time. Cortical cells were spherical in shape and had the activities of proliferation and steroid production in collagen gel matrix of this culture method. Aldosterone production in particular tended to be maintained much longer than in conventional monolayer culture. This method seems to provide a physiological environment in which to study the differentiation of cortical cells.