We previously reported the detection of a carbon-centered radical adduct of alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN) in the bile of rats acutely poisoned with Cr(VI) utilizing an electron spin resonance spin-trapping technique. These former studies suggested that the free radical metabolite was derived from a polyunsaturated fatty acid. The present studies were undertaken to further characterize this radical adduct and to determine whether its formation is associated with enhanced lipid peroxidation in vivo. This report demonstrates that electron spin resonance (ESR) spectra with hyperfine coupling constants aN of 15.71 G and of 2.90 G were present in bile from Cr(VI)-poisoned rats. We found out that virtually identical ESR spectra were obtained when authentic POBN-pentyl radical adducts generated from the reaction of POBN with either pentylhydrazine or linoleic or arachidonic acid with lipoxygenase were added to bile. The hyperfine coupling constants for the POBN-pentyl radical adducts added to bile were as follows: aN = 15.85 G and = 2.60 G for the reaction between pentylhydrazine and POBN; aN = 15.72 G and = 2.61 G for the reaction between arachidonic acid, lipoxygenase, and POBN; and aN = 15.85 G and = 2. 85 G for the reaction between linoleic acid, lipoxygenase, and POBN. In addition, the formation of this radical adduct was associated with lipid peroxidation as quantified by increases in F2-isoprostane levels in bile. These studies, therefore, provide additional evidence that acute Cr(VI) poisoning is associated with enhanced generation of F2-isoprostanes in vivo and tentatively identify the radical species that is produced as the POBN-pentyl radical adduct.