Manganese enhanced MRI (MEMRI) is an emerging technique for tracing neuronal pathways in vivo. However, manganese may leak into blood vessels or cerebrospinal fluid (CSF) after local injection and can be circulated to and taken up by brain regions that may not have connections to the targeted pathways. Comparing enhancement time courses after intranasal injection with intravenous infusion of MnCl(2) in rats, the early enhancements in the pituitary gland (Pit) and hippocampus indicate the contrasts in those regions in the olfactory tract-tracing experiment were caused by such systemic effects. Since the Pit has easy access to manganese from the blood and its signal is proportional to other brain regions after intravenous infusion, it was used as an internal reference for the systemic effects. Applying intensity normalization by the Pit signal to tract-tracing data from the olfactory bulb led to reduced contrast in the hippocampus. These results demonstrate that nonspecific enhancements in MEMRI tract-tracing studies may have to be taken into account and that normalization by the Pit signal can compensate these effects.