We conducted a study on the primary in vitro activation of T cells from non-sensitized mice by using hapten-conjugated Pam 212 cells (keratinocyte cell line). Furthermore, we attempted to develop a simple, quantitative in vitro test to assess the sensitizing potency of contact allergens and applied it to determine the stimulation index (SI) of various chemicals with known degrees of sensitizing potency. Monolayered Pam 212 cells were incubated with a variety of chemicals exhibiting allergenic potential. Washed and fixed T cells depleted of autoreactive T cells and macrophages from spleens of nonsensitized Balb/c mice were cocultured for 5 days with those monolayered Pam cells conjugated with chemicals. They were then harvested and restimulated with mitomycin-C-treated spleen cells conjugated with chemicals in 96-well culture plates to inhibit the proliferation of stimulator cells. We evaluated the sensitizing potency of the following chemicals: oxazolone, TNBS, DNFB and FITC (strong sensitizers); p-phenylendiamine (p-PD), nickel chloride and potassium dichromate (potent sensitizers); betamethasone and budesonide (corticosteroids), and methyl salicylate (MS) as an irritant. T cells sensitized in vitro with TNP-Pam cells and macrophages demonstrated antigen-specific proliferation when restimulated in vitro with mitomycin-C-treated TNP-spleen cells. Subcutaneous injection of these T cells induced contact sensitivity in vivo in an antigen-specific fashion. While T cells cocultured with TNP-3T3 could not be activated even in the presence of macrophages. The SI of strong sensitizers was about 4.00 and that of p-PD was 2.36. The SIs of budesonide, betamethasone and MS were 1.62, 0.98 and 1.21, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)