Manganese (Mn(2+))-enhanced MRI (MEMRI) is known to provide insight into functional and anatomical biology. However, this method, which uses Mn(2+) as a MRI-detectable contrast agent, has drawbacks such as the toxicity to cells beyond a certain level of Mn(2+). In this study, we attempt to determine a new method of ICV administration, the optimal concentration of administered Mn(2+) and the optimal MEMRI acquisition time following administration. Male Sprague-Dawley rats were used in the following experimental sessions: (1) intracerebroventricular (ICV) cannula implantation in the region of the cisterna magna, (2) serial dilution of MnCl(2) (20-80 mM), (3) ICV administration of MnCl(2) through the cannula, and (4) T(1)-weighted MRI measurements. We confirmed that cannula implantation in the region of the cisterna magna was a new ICV injection method for the administration of a contrast agent. The optimal concentration for MEMRI was 20/50 mM/µl of MnCl(2). The MEMRI data acquired at different time points indicate that most signal enhancement is maintained during 14-48 h after contrast agent injection, and 24 h was the optimal time to acquire images of the rat brain. The present study offers optimized parameters for contrast agent injection that would be a good basis for studies using MEMRI to research the rat brain.