Organotypic cultures, prepared from young rats, were used to investigate the neuroendocrine properties of anterior pituitary cells. Pituitary cells maintained the features of endocrine cells, up to 7 weeks in vitro. Secretory granules could be seen with electron microscopy, and cells contained immunocytochemically detectable levels of adenohypophyseal hormones. Significant levels of prolactin (PRL), growth hormone and luteinizing hormone were present in the culture media after several weeks in vitro and PRL release could be modulated by dopaminergic agonists or forskolin. The electrophysiological properties of pituitary cells were investigated with both intracellular and patch-clamp recordings after 2 to 7 weeks in vitro. Cellular resting membrane potentials were approximately -50 mV, and spontaneous or depolarization-induced action potentials were found in approximately 50% of cells. Records of voltage-dependent outward membrane currents showed that cells expressed functional voltage-gated channels. Cells remained responsive to hypothalamic neuropeptides, as shown by the outward membrane current triggered by thyrotropin-releasing hormone. Intracellularly injected Lucifer Yellow readily diffused between neighboring cells, suggesting the presence of gap junctions. These data confirm the viability of organotypic cultures of the anterior pituitary gland, and demonstrate that the characteristic properties of this excitable endocrine tissue are conserved. This neuroendocrine preparation is suitable for studying the mechanisms regulating cell-to-cell communication under conditions resembling the in vivo tissue organization.