The effect of iron, lactose and copper on manganese retention was studied in rats fed two diets. Thirty-six male albino rats (75 to 100 g) were allotted to six groups of six rats each. Three groups received a purified manganese-free dextrose-casein diet, and three groups received the same purified diet with 17% lactose added at the expense of dextrose. One group fed each of the above diets received either a manganese-free mineral mixture, the mineral mixture with 5 ppm supplemental Cu or the same mineral mixture with Fe removed. After 7 days on the diets, each rat was given, by gavage, 10 muCi of 54Mn activity as 54MnCl2 in a sodium acetate buffer. On the third day after dosing, the rats were sacrificed and samples of liver, kidneys, semitendinosus muscle, spleen and tibia were taken for stable and radioactive manganese analysis. Lactose added to the purified diet depressed 54Mn retention in all tissues studied. Lactose addition also decreased specific activities of the livers and kidneys but tended to increase stable manganese values. Copper apparently had little direct effect on 54Mn retention but tended to reduce the effect of lactose on 54Mn retention. Omission of Fe greatly increased 54Mn retention values in all tissues studied with or without added lactose. Fe omission also significantly increased the specific activities and stable Mn values of livers and the specific activity of kidneys. The results indicate that low dietary Fe may be a contributing factor to the increased manganese retention observed in this study and a previous study.