In activation-induced manganese-enhanced MRI (AIM-MRI) experiments, differential accumulation of Mn in activated and silent brain areas is generally assessed using T(1)-weighted images and quantified by the enhancement of signal intensity (SI), calculated with reference to SI before Mn administration or to SI of brain regions unaffected by the specific stimulus. However, SI enhancement can be unreliable when animals are removed from and reinserted into the magnet. We have developed an experimental protocol based on repeated intraperitoneal (i.p.) injections of Mn, quantitative determination of T(1), and coregistration of images to a rat brain atlas that allows absolute quantification of Mn concentration in selected brain areas. Results showed that interanimal variability of postcontrast T(1) values was very low (compared to the experimental error in T(1) determinations) allowing detection of differential regional Mn uptake in stimulated and unstimulated animals. In addition we have determined in vivo relaxivity of Mn in brain tissue and its frequency dependence.