Suspensions of isolated rat pituitary cells and gonadotroph- or lactotroph/somatotroph-enriched subpopulations obtained by unit gravity sedimentation were allowed to aggregate by constant gyrotory shaking, yielding aggregates of 100–150 μm. Within a few days, the aggregated pituitary cells became organized in a tissue-like configuration. There was no proliferation of mesenchymal cells. Glandular cells had a round to oval shape and formed specialized cell junctions. Areas of close apposition alternated with more dilated intercellular spaces. The different pituitary cell types retained their characteristic ultrastructural features and secretory granules, typical of the various cell types. Functional characteristics could be accurately studied in a superfusion system. LH, FSH, and PRL secretion showed very rapid on-off responses to nanomolar concentrations of the specific regulatory stimuli LHRH, TRH, vasoactive intestinal peptide, and dopamine. Aggregates remained highly responsive to LHRH even after 3 weeks in culture. The results indicate that isolated pituitary cells allowed to reaggregate in suspension culture form viable and functional multicellular structures which have maintained in vivo characteristics.