We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (.NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N-methyl-D-glucamine dithiocarbamate-ferrous iron, [(MGD)2/Fe], which binds to .NO and forms a water-soluble [(MGD)2/Fe-NO] complex. At 2 h after injection of the [(MGD)2/Fe] complex, a three-line EPR signal characteristic of the [(MGD)2/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD)2/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 microM, respectively. This 25-fold increase in .NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of .NO in mice. Administration of N-monomethyl-L-arginine (NMMA; 50 mg/kg) inhibited the ex vivo signal intensities of the [(MGD)2/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of 15N-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)2/Fe-14NO] complex and a two-line spectrum of the [(MGD)2/Fe-15NO] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected .NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible .NO synthase activities in living animals under physiological as well as pathophysiological conditions where .NO is overproduced.