The presence of Langerhans cells (LC) within the epidermis has been shown to be critical for inducing T-cell-mediated immune responses in the skin. The purpose of this study was to assess whether cells in the dermis can initiate T-cell-mediated delayed-type hypersensitivity responses in vivo. Initially, back skins from C3H mice were trypsinized to remove the epidermis. The dermis was enzymatically dispersed and filtered to obtain a cell suspension. However, dermal cells exposed to trypsin were contaminated with numerous disaggregated hair follicles. These hair follicles contained Ia+ cells (presumably LC), and upon haptenation in vitro with trinitrophenyl, initiated contact hypersensitivity reactions in vivo. We therefore used dispase in place of trypsin to prevent follicular disaggregation and to allow preparation of dermal cell suspensions free of hair follicles. These hair follicle-free dermal cells were haptenated with trinitrophenyl and injected intradermally. Elicitation of contact hypersensitivity by epicutaneous painting 6 d later revealed the mean +/- SEM incremental ear-swelling response to be 53 +/- 8 mm X 10(-3). In contrast, mice sensitized by injection with dermal cells depleted of Ia+ cells demonstrated only 10 +/- 1 mm X 10(-3) of ear swelling. Thus, like dendritic LC of the epidermis, perivascular dendritic Ia+ cells of the dermis are capable of initiating T-cell-mediated contact hypersensitivity in vivo and may be highly relevant for presentation of antigen to T cells trafficking through the dermis.