A recent report from our laboratory describes techniques for growing fetal murine endolymphatic sac (ELS) in vitro in tissue culture. The purpose of the present study is twofold: first, to determine whether the in vitro endolymphatic cells function in their artificial environment; and second, to begin to understand the nature of luminal cell function in vitro when separated from subepithelial connective tissue and blood. The endolymphatic sac was dissected from 18th gestational day fetal mouse otocysts and grown in DME tissue culture media for 7 days. Light microscopic sections of the endolymphatic sac were stained with Periodic Acid Schiff (PAS) reagent, alcian blue, Verhoff's elastin stain, and van Gieson's collagen stain to reveal deposits of glycogen as well as mucopolysaccharides, elastic fibers, and collagen. Controls for glycogen staining were prepared using the amylase enzyme. For electron microscopical evaluation, 'en bloc' staining was used, to confirm the location of cellular glycogen. Results indicate that in vitro luminal cells of the murine ELS are viable and show signs of functional activity with the markers used. The luminal substance and apical cytoplasm shows distinct purple metachromasia with toluidine blue and PAS-positive staining that disappears with amylase digestion. The ELS cells and luminal substance were negative for alcian blue at pH 1.0 and pH 2.5. These findings are similar to those seen in in vivo murine control sac specimens and demonstrate the ability of cultured sac cells to store glycogen and produce complex carbohydrates. The similarity in staining between the luminal substance and the cytoplasm of sac cells in all in vitro specimens suggests some secretory function by sac cells.