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W e studied glycoprotein content of human colonic goblet cells, using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). Using indirect immunofluorescence (IIF), we found that 17 of 23 anti-HCM MAbs stained some or all goblet cells of normal human colonic mucosa. We observed a variety of cellular staining patterns, including (a) diffuse (homogeneous) staining of intracellular mucin, (b) speckled (inhomogeneous) staining of mucin droplets, (c) peripheral staining of intracellular droplets, (d) cytoplasmic staining of goblet cells, and (e) apical (luminal) surface staining. Staining patterns were not associated with particular HCM species. In addition to variable patterns of IIF within individual cells, anti-HCM MAbs varied in the proportion of goblet cells stained. Some MAbs stained all goblet cells, while others stained a limited number of goblet cells. Although each goblet cell contained more than one type mucin, HCM species III, and IV and V appeared to exist in mutually exclusive goblet cell populations and it was possible to define at least seven subpopulations of goblet cells in colonic mucosa by their content of various combinations of HCM species. Anti-HCM MAbs stained goblet cells from other sites within the gastrointestinal tract to a varying extent. Anti-HCM MAbs also showed extensive cross-reactivity with rodent, rabbit, and monkey colonic mucosa. However, several anti-HCM MAbs stained only human colonic mucosa. These data show that human colonic mucosa contains discrete subpopulations of goblet cells that produce distinctive combinations of specific mucin glycoprotein species.