MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8(+) MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon gamma (IFNgamma), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (-/-) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNgamma + VV-IL-2, VV-IFNgamma + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNgamma had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (-/-) mice demonstrated that mice that are IL-2 -/- and IL-7 receptor -/- produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 -/-, IL-10 -/- and IFNgamma -/- mice were marginally improved and responses to M-FP in IL-4 -/- and tumour necrosis factor receptor 2 -/- mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1(+) P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone.