To study the nature of reticular epithelial (REp) cells and their role in the specific microenvironments of the chicken bursa and thymus, a method was developed for the in vitro culture of purified preparations of these cells. For comparison, similar cultures of splenic adherent cells were also performed. REp cell-rich bursa medullary follicles and mildly trypsinized thymic fragments were X-irradiated (850 rad) to eliminate remaining lymphocytes and transferred to culture flasks. In bursal cultures, after 2-4 days incubation the basement membrane (BM) encapsulating the follicles disrupted and the immediately underlying epithelial cells grew out as a monolayer. By 10 days, REp cells at the periphery developed cytoplasmic processes; occasionally these cells appeared to "bud-off" and grow as isolated dendritic cells. Thymic REp cells were generally slower to proliferate but formed a monolayer by 10-14 days. Splenic adherent cells developed extensive growth within 4 days. REp cells were distinguished from fibroblasts, when present, morphologically and by their limited phagocytic ability. The former were also periodic acid-Sciffs reagent (PAS)-positive and produced reticulin granules. Bursal REp cells were also positive for a gut-associated mucin, but this may have been bound in vivo prior to culture. Neither T nor B lymphocyte-specific antigens were detectable on the cultured REp cells or splenic adherent cells, but they were all rich in cytoplasmic actin. A major feature of REp cells to emerge in this study was the obvious presence of subpopulations of these cells, which raises important questions as to their exact nature and lineage. The accompanying paper details the ability of the bursal and thymic REp cell cultures to induce B-or T-lymphocyte differentiation, respectively, in vitro.