The biologically active metabolite of vitamin D3, 1 alpha, 25-dihydroxycholecalciferol (1,25-(OH)2D3), inhibited the metabolic cooperation between cocultivated Chinese hamster V79 cells that are either susceptible or resistant to the cytotoxic effect of 6-thioguanine (TG). This inhibition was characterized by an increased survival of TG-resistant cells when they are cocultured with susceptible cells in the presence of TG. TG-resistant cells in the absence of susceptible cells and after treatment with TG yielded a cloning efficiency of about 90%. Cocultivation of these cells with the susceptible cells reduced this cloning efficiency to about 30%. At 0.5 microgram/ml, 1,25-(OH)2D3 restored the cloning efficiency of the TG-resistant cells in the cocultures to 62%. Phorbol 12-myristate 13-acetate (PMA), a known inhibitor of metabolic cooperation, at 0.5 microgram/ml restored the cloning efficiency to a similar extent. Vitamin D3, 24,25-dihydroxycholecalciferol (24,25-(OH)2D3, and phorbol were ineffective in inhibiting this metabolic cooperation. 1,25-(OH)2D3, unlike PMA, did not inhibit the binding of [3H]phorbol 12,13-dibutyrate (PDBu) to its cellular receptors in either the TG-susceptible or resistant cells. These results indicate that 1,25-(OH)2D3 resembles tumor-promoting agents such as PMA in its capacity to inhibit metabolic cooperation, but this capacity is not mediated through phorbol diester receptors.