A series of human-human hybridomas derived from a single fusion of UC 729-6 with lymph node lymphocytes were examined for the type and nature of macromolecules synthesized and secreted. One hybrid, VLN3G2, secreted fourfold higher IgG than that present in the cytoplasm over 4 days of growth, while the IgM distribution was opposite to that of IgG. VLN5C7, contrary to VLN3G2, contained several-fold more cytoplasmic IgG as well as IgM than the amounts secreted over the same period of time. Of the secreted IgG and IgM by both of these hybridomas, only the IgG showed immunoreactivity against target A431 cell surface antigen(s). Another hybridoma, termed VLN1H12, secreted immunoreactive IgM against target A431 cells, but no detectable IgG. Cytoplasmic proteins prepared by repeated freeze-thaw of the hybridoma cells, membrane proteins obtained by NP-40 extraction of the cell membrane, and secreted proteins present in the supernates of the various hybridomas were assayed by an enzyme-linked immunoassay (ELISA), to understand the discrepancy observed in the immunoglobulins of the cellular and extracellular compartments. The parental UC 729-6 cell line used in these cell fusions produced only trace amounts of immunologically inactive IgM and no detectable IgG. Molecular sieving column chromatography of these hybridoma supernates suggested the presence of intact IgG and IgM molecules and the absence of free heavy chains or hybrid antibodies containing both mu and gamma heavy chains. Intrinsic labeling of VLN3G2 hybridoma cells with 35S-methionine demonstrated the presence of not only a nonimmunoglobulin protein but also a small molecular weight protein-A-binding polypeptide in the culture supernatant. 35S-methionine-incorporated IgG and IgM antibodies, isolated from spent media, cytoplasm, and cell membranes of VLN3G2, also showed binding to protein-A-bearing bacteria. In conclusion, the differences observed in the amounts of secreted MAbs by the human-human hybridomas were not due to the decreased synthesis of these molecules.