This study was performed to investigate the effect of Matrigel, a reconstituted basement membrane, on the expression of the anterior pituitary hormones in culture. Rat pituitary cells cultured for 6 days on Matrigel showed 3-dimensional, lobular structures with connecting cells while those on plastic showed flat, polygonal cells forming a monolayer. Western blot analysis showed that prolactin (PRL) content in the anterior pituitary cells was higher compared to those cultured on plastic. In comparison, TSH expression was not increased in cultures on Matrigel. The total cell number and the proportion of fibroblasts was decreased. These results suggested that Matrigel is a useful culture substrate for the enhanced expression of PRL but not for TSH. Further studies are needed in order to find a useful culture substrate for TSH cells.