Food irradiation is a preservation method that has been approved by the European Union for a limited number of foods or food products and within a limited range of doses. European Community legislation states that all foods or food ingredients treated in this manner and authorized for sale in the European Union must be clearly labeled, and that market-level inspections must be carried out to ensure compliance with this regulation. To improve detection of irradiated foods, we evaluated the efficacy of electron spin resonance (ESR) spectroscopy for the identification of seafood that had been subjected to irradiation. Three fish species were tested: gilthead sea bream, cod, and swordfish. For each species, 18 samples of flesh-containing bone were analyzed after either irradiation at 0.5, 1, or 3 kGy or no irradiation (control). Induced radicals in irradiated samples produced distinct ESR signals that allowed differentiation from non-irradiated samples. Within the dose range tested, a linear dose-response relationship was observed (R(2) > 0.94). The gilthead sea bream displayed the highest radiosensitivity. The analytical procedure was validated and successfully used for the routine monitoring of locally marketed seafood. The main aims of the study were to develop a reliable tool for the enforcement of the proper labeling of irradiated foods and to obtain preliminary data on compliance with current laws in the Italian market.