Effects of antibody feedback regulation on T helper (Th) cell functions required in plaque-forming cell (PFC) responses were studied in vitro. Using a sensitive functional assay, it was found that serum from immunized mice did not inhibit sheep erythrocyte (SRBC)-, or keyhole limpet hemocyanin-specific Th cell activation. SRBC-"pulsed" splenic adherent cells activated Th cells more efficiently when pulsed in the presence of anti-SRBC antiserum. PFC responses against, 2,4,6-trinitrophenyl (TNP) coupled to soluble carriers were studied under conditions in which linkage between hapten and the priming carrier was either required or not. Under both conditions, anti-carrier antiserum had an enhancing or no effect; only anti-hapten antiserum or monoclonal anti-hapten antibody were inhibitory. Thus, these results did show that antibody feedback was essentially hapten-specific and did not interfere with Th cell functions, except, possibly, that anti-hapten antibody interfered with linked cooperation at the level of hapten-B cell interaction, in addition to its inhibitory effect detectable under conditions of unlinked cooperation. Since SRBC-pulsed, adherent cells were very inefficient in stimulating anti-SRBC PFC responses, the results further suggested that phagocytosed antigen could only be recognized by Th cells but not by B cells. Finally, by attempting to further study hapten-B cell interactions, no PFC responses against TNP coupled to soluble carriers could be generated by substituting for Th cells with different types of secondary mixed leukocyte culture supernatant in cultures with pure surface Ig-positive cells. Only anti-SRBC PFC responses were generated in such cultures.