We investigated the relationship between transforming growth factor‐β (TGF‐β)‐secreting T‐regulatory (Tr) cells and anti‐B16 melanoma immunity, and studied the association of early cytokines expressed at tumour sites with the generation of Tr cells. A large number of CD4⁺ Tr cells producing interleukin (IL)‐4, IL‐10 and TGF‐β accumulated with functionally depressed CD8⁺ cytotoxic T lymphocytes (CTLs) at tumour sites on day 20 after subcutaneous (s.c.) inoculation of B16 tumour cells. Tr cells consisted of two populations, which were termed T helper 3 (Th3) and Tr1 cells. B16‐infiltrating Tr cells strongly inhibited the generation of B16‐specific T helper 1 (Th1) cells in a TGF‐β‐dependent manner and were assumed to suppress effective generation of CTLs. In addition, B16 cells markedly progressed in mice transferred adoptively by the cultured B16‐infiltrating Tr cells compared with untreated mice. The capacity of these Tr cells to produce TGF‐β was hampered by neutralizing anti‐IL‐10 and partly anti‐IL‐4 monoclonal antibodies (mAbs) injected intralesionally during the early development of B16 tumours, and this treatment markedly attenuated B16 growth. Furthermore, a lesional injection of recombinant mouse IL‐10 at an early tumour site resulted in the vigorous progression of B16 tumours. These results provide evidence that Tr cells, belonging to the T helper 3/T‐regulatory 1 (Th3/Tr1) type, are activated in B16‐bearing hosts under the influence of T helper 2 (Th2) cytokines, mainly IL‐10 (produced at early tumour lesions), and that this regulatory T‐cell population functions as a suppressor of anti‐B16 immunity.