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This study aimed to develop the method for determination of the ultra-small superparamagnetic iron oxide nanoparticle (USPION)-originated iron (UOI) in the tissues of rats on the basis of the magnetic characteristics (MC) in the liver, left heart ventricle (LHV), kidneys, aorta and blood of Wistar-Kyoto (WKY). Rats were treated intravenously by USPIONs dispersed in saline (transmission electron microscope (TEM) mean size ~30 nm, hydrodynamic size ~51 nm, nominal iron content 1 mg Fe/mL) at the low iron dose of 1 mg/kg. MC in the form of the mass magnetisation (M) versus the magnetic field (H) curves and temperature dependences of M (determined using the SQUID magnetometer), histochemical determination of iron (by Perl’s method) and USPION-induced superoxide production (by lucigenin-enhanced chemiluminescence) were investigated 100 min post-infusion. USPIONs significantly elevated superoxide production in the liver, LHV, kidney and aorta vs. the control group. Histochemical staining confirmed the presence of iron in all solid biological samples, however, this method was not suitable to unequivocally confirm the presence of UOI. We improved the SQUID magnetometric method and sample preparation to allow the determination of UOI by measurements of the MC of the tissues at 300 K in solid and liquid samples. The presence of the UOI was confirmed in all the tissues investigated in USPIONs-treated rats. The greatest levels were found in blood and lower amounts in the aorta, liver, LHV and kidneys. In conclusion, we have improved SQUID-magnetometric method to make it suitable for detection of low amounts of UOI in blood and tissues of rats.