This study was designed to establish an in vitro model system in human hormone-producing pituitary adenoma which is analogous to the in vivo cellular environment. Mechanically dispersed cells composed of single cells and aggregated cells from 9 pituitary adenomas (5 GH-producing adenomas and 4 prolactinomas) were cultured on microporous membrane coated with basement membrane extract. Not only single cells but also large aggregated cells which usually float in the medium when seeded on ordinary plastic flasks were morphologically flattened and firmly attached to coated microporous membrane within a few days under the control of seeding medium volume. In both types of adenoma, surviving aggregated adenoma cells revealed well-preserved hormonal activity with regard to both cell morphology and hormone release after 6 m in culture and no undifferentiation of cell characteristics was observed. It is concluded that this culture method may further aid the investigation of in vitro cellular structure and function in human pituitary adenoma under conditions which closely mimic the in vivo cellular environment.