We have compared the size, the binding to Concanavalin A (Con A), and the affinity for monoclonal antibody 1116NS-199 (Mab 19-9) among CA 19-9 molecules from sera of cystic fibrosis (CF) and pancreatic carcinoma patients and from sputum extracts. CA 19-9 molecules of two different sizes were found in all types of specimens by Sepharose 4B chromatography. While the smaller CA 19-9 molecule was predominant in CF patient sera, the larger molecule was associated with most of the sera from patients with pancreatic carcinoma. The majority of the sputum extracts contained the larger CA 19-9 molecule. All CA 19-9 molecules studied by Con A chromatography did not appear to bind to Con A, and almost 100% were found in the nonreactive fraction. The CA 19-9 molecules from sera of either CF or pancreatic carcinoma patients exhibited variable affinities for Mab 19-9, some approaching that of the standard curve but many also having lower affinities. The lowest affinity was displayed by CA 19-9 molecules from the sputum extract. It appears that development of more specific assays for CF and for carcinoma is possible if the correct CA 19-9 molecule is selected for antibody preparation and for use as standards.