The characterization of a mucus-producing human cell line (HC-84) derived from a colon carcinoma and its response to in vitro reserpine treatment is reported. Mucous granules were demonstrated within these cells on the basis of electron microscopic examination and incorporation of [3H]glucosamine with subsequent autoradiographic analysis. Fluorographic analysis of total HC-84 cell protein after incubation with [3H]glucosamine indicated that the majority of tritium was incorporated into two proteins with molecular weights of 115 and 120 kD. When total HC-84 protein was subjected to immuno-blot analysis utilizing rabbit antibody against human intestinal mucus, only these two proteins (115K and 120K) reacted positively, indicating a direct correlation between [3H]glucosamine incorporation and mucus production. Immunofluorescence localization of mucus within HC-84 cells utilizing this same antibody resulted in a punctate pattern of fluorescence within the cytoplasm. Treatment of HC-84 cells with 30 microM reserpine for 7 days resulted in a three-fold increase in mucus production compared with controls. There was also a concomitant loss of a 30K calmodulin-binding protein in cells treated with reserpine. These cells represent a useful system for studying the effect of reserpine on the processes of mucus synthesis and secretion.