The ability of the ESR technique based on diethyldithiocarbamate (DETC) administration was studied as a suitable method to assess NO generation in vivo. The technique was successfully employed to measure NO generation after LPS treatment. DETC2-Fe-NO adducts were detected in liver homogenates of iron overloaded animals. When iron was administered to the animals simultaneously with LPS, NO-dependent signal increased 122%, but the content of NO2- and NO3- in sera was significantly lower (44%) as compared to LPS-treated rats. Iron dextran administration was responsible for a three-fold increase in the DETC2-Fe-NO content in non-LPS treated rats, while NOS activity and sera NO2- and NO3- levels remained unaffected. The adduct generation rate by a chemical NO-source was recorded in the presence of either control or iron overloaded homogenates supplemented with DETC in vivo. The exposure of liver homogenates to NO was performed either by the addition of 1 mM SNAP as NO donor or infusing an aqueous NO solution. In the presence of iron overloaded samples the adduct generation rate was 3.8-4.4-fold higher than in the presence of control samples. This effect restricts the applicability of the method to experimental conditions where iron levels remain constant, therefore it is not suitable for NO generation studies in experimental models where animals were subjected to iron overload.