Melanoma represents a significant challenge in cancer treatment due to the high drug resistance of melanomas and the patient mortality rate. This study presents data indicating that nanomolar concentrations of the hormonally active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)(2)D3], its non-calcemic analogues 20S-hydroxyvitamin D3 and 21-hydroxypregnacalciferol, as well as the low-calcemic synthetic analog calcipotriol, modulate the efficacy of the anticancer drugs cisplatin and dacarbazine. It was observed that vitamin D analogs sensitized melanoma A375 cells to hydrogen peroxide used as an inducer of oxidative stress. On the other hand, only 1 alpha,25(OH)(2)D3 resulted in a minor, but significant effect on the proliferation of melanoma cells treated simultaneously with dacarbazine, but not cisplatin. Notably, cisplatin (300 mu M) exhibited a higher overall antiproliferative activity than dacarbazine. Cisplatin treatment of melanoma cells resulted in an induction of apoptosis as demonstrated by flow cytometry (accumulation of cells at the subG(1) phase of the cell cycle), whereas dacarbazine caused G(1)/G(0) cell cycle arrest, with the effects being improved by pre-treatment with vitamin D analogs. Treatment with cisplatin resulted in an initial increase in the level of reactive oxygen species (ROS). Dacarbazine caused transient stimulation of ROS levels and the mitochondrial membrane potential (Delta psi(m)) (after 1 or 3 h of treatment, respectively), but the effect was not detectable following prolonged (24 h) incubation with the drug. Vitamin D exhibited modulatory effects on the cells treated with dacarbazine, decreasing the half maximal inhibitory concentration (IC50) for the drug, stimulating G(1)/G(0) arrest and causing a marked decrease in Delta psi(m). Finally, cisplatin, dacarbazine and 1 alpha,25(OH)(2)D3 displayed modulatory effects on the expression of ROS and vitamin D-associated genes in the melanoma A375 cells. In conclusion, nanomolar concentrations of 1,25(OH)(2)D-3 only had minor effects on the proliferation of melanoma cells treated with dacarbazine, decreasing the relative IC50 value. However, co-treatment with vitamin D analogs resulted in the modulation of cell cycle and ROS responses, and affected gene expression, suggesting possible crosstalk between the signaling pathways of vitamin D and the anticancer drugs used in this study.