A major emphasis in cancer therapy research is finding mechanisms to enhance the effectiveness of clinically used chemotherapeutic agents. In this report, we show the effects of direct NO exposure or NO delivery agents such as NONOate NO donors, DEA/NO ((C2H5)2N[N(O)NO]-Na+) and PAPA/ NO (NH2(C3H6)(N[N(O)NO]C3H7)), or S-nitrosothiol NO donors (GSNO, S-nitrosoglutathione, and SNAP, S-nitroso-N-acetylpenicillamine) on the cytotoxicity of cisplatin with Chinese hamster V79 lung fibroblast cells. Cells pretreated with bolus NO or NO delivered from NONOate NO donors were markedly sensitized to subsequent cisplatin treatment, whereas S-nitrosothiol NO donors exerted little effect. The enhancement in cisplatin cytotoxicity from pretreatment with DEA/NO and PAPA/ NO persisted for approximately 180 and 240 min, respectively; thereafter cytotoxicity returned to a level consistent with cisplatin treatment alone. Pretreatment of cells with GSNO or SNAP did not enhance cisplatin cytotoxity. To discern why there were differential effects among the different NO donors, formation of NO over the time course of the experiment was assessed by the nitrosation of 2,3-diaminonaphthylene. Bolus NO, DEA/NO, and PAPA/NO produced more reactive nitrogen oxide species (RNOS) than did treatment with GSNO or SNAP. Previously reported electrochemical studies revealed that temporal NO concentrations measured from DEA/NO and PAPA/NO (1 mM) were greater than 5 microM. It appears that the flux of NO, as well as the amount of RNOS, is important in the NO-mediated enhancement of cisplatin cytotoxicity. Our results demonstrate the importance of NO delivery systems in the enhancement of cisplatin cytotoxicity and may provide insights into strategies for participation of NO donors and nitric oxide synthase with cisplatin therapy.