The adjuvant activities of four chemically similar, but physicochemically different nonionic surface-active agents called pluronic polyols F 68, L 31, L 101 and L 121 were studied. These four agents were tested in mice using an experimental model developed for studying the adjuvant activity of the cationic surface-active agent dimethyl dioctadecyl ammonium bromide (DDA). L 121 and DDA enhanced the primary antibody response to sheep red blood cells (SRBC) while F 68, L 31 and L 101 suppressed this response. The secondary humoral response to SRBC was enhanced by the polyol L 121 while the secondary response to dinitrophenylated bovine serum albumin (DNP22-BSA) was enhanced by both L 121 and L 101. DDA and the polyol L 101 were very effective adjuvants for induction of delayed-type hypersensitivity (DTH) to SRBC and DNP22-BSA after intracutaneous immunization of mice with a mixture of antigen and adjuvant. Since the four pluronic polyols were composed of identical chemical constituents, we proposed that difference in their activities as adjuvants were due to variation in their physicochemical properties. A correlation was found between a physicochemical parameter, the hydrophilelipophile balance (HLB), and the adjuvant activities of the pluronic polyols and several other types of nonionic surface-active agents. The agents which were strong adjuvants all had HLB values within a narrow range which classified them as spreading agents.