The specificity of a cell-mediated immune rsponse in mice artifially immunized with T. (S.) dionisii or T. (S.) vespertilionis was studied using as indicator the delayed hypersensitivity footpad skin test. No delayed response significantly greater than that occurring in the footpad challenged with saline resulted from challenging non-immune mice with either trypanosomal or medium antigens, nor did any delayed response occur in mice immunized with FCA [Freunds complete adjuvant] and challenged with either trypanosomal antigen or medium. Strong, highly significant (P < 0.05) delayed responses occurred in mice immunized with trypanosomal antigen or medium and challenged with homologous antigen. Slight but significant delayed cross-reactions occurred in mice immunized with T. dionisii and challenged with T. vespertilionis and in the reciprocal test, indicating that the 2 spp. possessed some shared antigens. Similar cross-reactions occurred in mice immunized with T. vespertilionis and challenged with medium, in the reciprocal test and in mice immunized with medium and challenged with T. dionisii; these were probably caused by contamination of the trypanosomal antigen by endocytozed medium. Mice immunized with T. dionisii and challenged with medium did not give a delayed response, possibly because T. dionisii, a smaller organism in vitro than T. vespertilionis, contained traces of medium insufficient to produce an immune response; mice immunized with medium in the reverse reaction could be sensitized so that traces of medium in the T. dionisii challenge antigen could produce a response. An antigenic difference between T. dionisii and T. vespertilionis, although incomplete, seemed evident.