We studied the specificity of protection conferred by Toxoplasma gondii immune lymphocytes and their supernatants on infected hamster kidney cells, using Besnoitia jellisoni immune lymphocytes as a nonspecific control. The intracellular growth of the organisms was measured by [3H]uracil incorporation, and inhibition of multiplication was used as a measurement of immunity. Although the immune lymphocytes restricted principally the multiplication of homologous organisms, partial protection, expressed against the heterologous organism, was found. This was true for either parasite with intact lymphocytes or their supernatants. Exposure of immune lymphocytes to antigen for 18 to 24 h and treatment of kidney cells with supernatant fluids for 18 to 24 h were required for maximal protection. The specific protective mediator in supernatants of immune lymphocytes was characterized by dialysis as having a molecular weight between 3,000 and 12,000 and was found in the 3,000 to 5,000 peak after Sephadex G-50 chromatography. Nonspecific protective activity was greater than 12,000 by dialysis; it chromatographed in the excluded peak, measuring over 43,000, and was destroyed by exposure to pH 2. In vitro production of lymphokines from toxoplasma immune lymphocytes was first detected 7 to 10 days after vaccination of hamsters. At about the same time, hamsters began to resist challenge infection with the pathogenic RH strain of T. gondii and were able to prevent its multiplication in lungs, liver, spleen, and the subcutaneous infection site. The expression of tissue immunity and the production of toxoplasma-immune lymphokines appear to be time-related events.