The adjuvant activity of mycobacterial RNA and synthetic polynucleotides for the induction of delayed hypersensitivity to PPD was determined. It was shown that when mycobacterial RNA or synthetic polynucleotides are injected together with purified protein derivative (PPD), delayed hypersensitivity to PPD developed as compared to no detectable delayed response when PPD was administered alone without adjuvant into guinea pigs. Four different criteria were employed to detect delayed hypersensitivity responses. These were the time, appearance, and magnitude of dermal reactions, histologic examination of dermal sections, passive transfer of sensitivity with sensitized spleen cells and the elaboration of migration inhibitory factor (MIF) by sensitized spleen cells. When synthetic polynucleotides were used as adjuvants and were injected into guinea pigs in combination with PPD, dermal reactions as well ad MIF assays gave evidence that these animals exhibited delayed-type hypersensitivity. Poly U alone exhibited adjuvant activity for induction of delayed hypersensitivity to PPD. Trypsin and pronase treatment did not affect the adjuvant activity of mycobacterial RNA whereas KOH treatment completely abolished any adjuvant effect, suggesting that ribosomal protein did not contribute to the adjuvant characteristics of mycobacterial RNA. Titration experiments indicated that the adjuvant activity of mycobacterial RNA was greater than that of poly A:U.