A mouse-model was used to investigate the effect of methotrexate (MTX) and folinic acid on accumulation of iron in young growing mice. Four equal groups of Balb/c young male mice were treated (subcutaneously) with either MTX, or folinic acid, or MTX plus folinic acid, or physiological saline on every second day. After 3 weeks of treatment, liver, spleen, kidney, small intestine, brain, skeletal muscle and heart were removed and analyzed for iron contents using a spectrophotometric method. When the mean values of iron in liver of four groups were compared using one way ANOVA followed by Tukey's HSD test, the group receiving MTX alone was found to have significantly (p=0.004) more accumulation of iron in liver. The group receiving MTX plus folinic acid had iron accumulation in the liver similar to the placebo group. However, the mean values of iron in brain, kidney, small intestine, skeletal muscle, heart and spleen in all the groups, were not found to be statistically different. The data indicate that MTX shifts iron from being utilized in hemoglobin synthesis to liver stores. Folinic acid administration 8 h post-MTX, however, prevents this shift of iron to liver. Decreased levels of iron in plasma in mice treated with MTX alone suggest decreased availability of iron to other tissues for their normal growth and development.