AbstractDelayed‐type hypersensitivity (DTH) is an in vivo assay of cell‐mediated immune function. DTH reactions are often divided into two phases: the sensitization phase, referring to the initial immunization with specific antigen, and the efferent or challenge phase of the DTH response, which usually follows 6 to 14 days after sensitization. This unit details the protocol for stimulating DTH responses in mice to the O‐succinimide ester of the hapten 4‐hydroxy‐3‐nitrophenyl acetyl (abbreviated NP‐O‐Su).