To gain a better understanding of the possible mechanisms by which a stable form of ascorbate, ascorbic acid 2-glucoside (AA-2G), as an ascorbate source, augments antibody responses, we examined whether AA-2G enhances the anti-sheep-red-blood-cell (SRBC) plaque-forming cell (PFC) responses elicited with distinct interleukins that provide signals for B-cell proliferation and differentiation in cultured murine T-cell-depleted splenocytes. The anti-SRBC PFC responses were markedly reduced by T-cell depletion; and additions of the concanavalin A-stimulated murine splenocytes supernatant (CAS) or interleukin (IL)-1beta, IL-2, IL-5, IL-4 or IL-6 to the culture limitedly restored the immune responses. AA-2G synergistically stimulated the anti-SRBC PFC responses in the presence of IL-1beta-, IL-2, IL-5 or CAS, IL-1beta among these cytokines being most highly affected. However, it failed to enhance the PFC responses elicited by IL-4 or IL-6. Repeated additions of ascorbic acid (AsA) during experimental periods could also produced the enhancing effect, but a single addition of the vitamin did not, because of its instability in the medium. It was shown that exposure to IL-1beta, IL-2 or IL-5 must be done at early times after antigen stimulation of the cells to support their optimal responses and that AsA exerted its effect on day 2 and day 3 after the start of culture. These results suggest that AsA may up-regulate the in vitro IgM antibody responses in a cytokine-dependent manner.