Colloidal dispersions of monodispersed and high-crystalline magnetite nanoparticles have been used to establish a relationship between magnetic properties and magnetic resonance (MR) relaxometric parameters in vitro. Magnetite nanoparticles with diameters between 4 and 14 nm were synthesized by thermal decomposition of Fe(acac)3 in different organic solvents and transformed to hydrophilic by changing oleic acid for dimercaptosuccinic acid (DMSA). A final treatment in alkaline water was critical to make the suspension stable at pH 7 with xi-potential values of -45 mV and hydrodynamic sizes as low as 50 nm. Samples showed superparamagnetic behavior at room temperature, which is an important parameter for biomedical applications. Susceptibility increased with both particle and aggregate size, and for particles larger than 9 nm, the aggregate size was the key factor controlling the susceptibility. Relaxivity values followed the same trend as the suspension susceptibilities, indicating that the aggregate size is an important factor above a certain particle size governing the proton relaxation times. The highest relaxivity value, r(2) = 317 s(-1) mM(-1), much higher than those for commercial contrast agents with similar hydrodynamic size, was obtained for a suspension consisting of 9 nm particles and 70 mu of hydrodynamic size, and it was assigned to the higher particle crystallinity in comparison to particles prepared by coprecipitation. Therefore, it can be concluded that in addition to the sample crystallinity, both particle size and aggregate size should be considered in order to explain the magnetic and relaxivity values of a suspension.