The objective of the current study was to evaluate increasing levels of manganese hydroxychloride (MHC) in 45-wk-old white leghorn laying hens, using yolk and shell manganese (Mn) content as a potential marker for Mn concentration. A total of 80, 45-wk-old white leghorns were assigned to 6 dietary treatments, each consisting of 14 individually caged laying hens, with the exception of the reference diet containing 10 individually caged laying hens. The experiment consisted of a reference diet that contained 70 ppm of supplemental inorganic Mn in the form of Mn oxide and 5 experimental treatments each containing 0, 15, 30, 60, and 90 ppm supplemental MHC. Experimental birds were subjected to a 21 D depletion phase in which no supplemental Mn was included in the diet; however, during this time reference fed birds were fed the control diet (70 ppm Mn). After the 21 D depletion phase, the depleted birds were fed experimental diets for a 35 D evaluation period. Yolk and shell Mn content were analyzed at the end of the depletion phase and during the experimental phase on day 5, 10, 15, 25, and 35. During the experimental phase, Mn was replenished in the yolk and shell in all experimental treatments containing supplemental Mn; however, dose and time impacted the rate of replenishment. The yolk tended to be more sensitive to variations in Mn level as increases in Mn inclusion significantly (P < 0.05) increased concentration. These data demonstrate the ability to deplete and replenish Mn, and the use of egg yolk Mn concentration as measurement for determining changes in dietary Mn. At the conclusion of the experiment at 35 D, 60 ppm of Mn hydroxychloride seemed to be adequate in replenishing Mn to the level of the reference.