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AC magnetic-field-induced heating, cytotoxicity, and bio-related physical properties of two kinds of spinel ferrite nanoparticles, soft (NiFe2O4) and hard (CoFe2O4), with different mean particle sizes were investigated in this paper to confirm the effectiveness for an in vivo magnetic nanoparticle hyperthermia agent in biomedicine. AC magnetically induced heating temperature of the nanoparticles measured both in a solid and an agar state at different applied magnetic fields and frequencies clarified that the maximum heating temperature of NiFe2O4 nanoparticles is much higher than that of CoFe2O4 nanoparticles. In addition, it was demonstrated that solid-state NiFe2O4 nanoparticles with 24.8 and 35 nm mean particle size exhibited a promisingly high heating temperature (21.5degC-45degC) for a hyperthermia agent in the physiologically tolerable range of the ac magnetic field with less than 50 kHz of applied frequency. According to the magnetic and physical analysis results, the superior ac magnetically induced heating performance of NiFe2O4 nanoparticles was primarily due to their higher magnetic susceptibility (permeability) that directly induces a larger magnetic minor hysteresis loop area at the low magnetic field. Cytotoxicity test results, quantitatively estimated by methylthiazol tetrazolium bromide test method, verified that uncoated NiFe2O4, chitosan-coated NiFe2O4, and CoFe2O4 showed a noncytotoxicity, which is clinically suitable for a hyperthermia agent application.