Regulatory T (Treg) cells suppress contact hypersensitivity (CHS) responses, but the dynamics, mode, and site of their action is not well characterized. We studied forkhead box P3+ (Foxp3+) Treg cells during the CHS response in conditional Foxp3 knockout depletion of regulatory T cell (DEREG) mice, where Foxp3+ cells can be transiently deleted by diphtheria toxin. The mice were sensitized and challenged with oxazolone, and Foxp3+ cells were depleted either during sensitization or elicitation. Treg cell depletion before sensitization led to significantly exacerbated and prolonged CHS responses. In contrast, depleting Treg cells during elicitation had no effect on the 24-hour response, but the response was significantly prolonged. In wild-type mice, the gradual resolution of the CHS response was accompanied by a similarly gradual accumulation of Foxp3+ Treg cells relative to T effector cells in the skin. This effect was not as marked in the Treg cell-depleted mice, suggesting that the skin is an important site of Treg cell activities during the resolution phase. Together, our results show that endogenous Foxp3+ Treg cell function is important during the sensitization and resolution phases, but their depletion just before elicitation does not have an effect on the CHS response during the first 24 hours after elicitation.