Basement membrane, a thin extracellular matrix, promotes tissue integrity and differentiated phenotype. This study was performed in order to investigate the effect of basement membrane components on adrenocorticotropin (ACTH) synthesis and to observe its relationship with cell morphology. Rat anterior pituitary cells were cultured on plastic culture plate coated with either Matrigel or poly-D-lysine. Phase-contrast microscopy and scanning electron microscopy showed that cells cultured on Matrigel appeared as a three-dimensional glandular-like cell aggregate, while those cultured on plastic showed a flat, confluent monolayer. Reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis revealed that ACTH synthesis in the Matrigel culture was not significantly different from that in the plastic culture. Our results suggest that the relationship between the morphological changes caused by cell-substrate interaction and pituitary hormone synthesis does not exist in all pituitary cell types and that other factors associated with cell-substrate interaction influence the hormone synthesis of some pituitary cells.