Folliculo-stellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. FS cells connect to each other not only by mechanical means, but also by gap junctional cell-to-cell communication. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture markedly change their shape, and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. Morphological and functional changes in cells are believed to be partly modified by matricrine signaling, by which ECM components function as cellular signals. In the present study, we examined whether gap junction formation between FS cells is affected by matricrine cues. A cell sorter was used to isolate FS cells from male S100b-GFP rat anterior pituitary for primary culture. We observed that mRNA and protein levels of connexin 43 in gap junction channels were clearly higher in the presence of laminin. In addition, we confirmed the formation of gap junctions between FS cells in primary culture by electron microscopy. Interestingly, we also observed that FS cells in the presence of laminin displayed well-developed rough endoplasmic reticulum and Golgi apparatus. Our findings suggest that, in anterior pituitary gland, FS cells may facilitate functional roles such as gap junctional cell-to-cell communication by matricrine signaling.