Unprimed (CBA X C57BL/6)F1 lymph node T cells were transferred with sheep erythrocytes (SRC) into heavily irradiated F1 or parental strain mice and recovered from thoracic duct lymph or spleens of the recipients 5 days later. To study their helper function, the harvested F1 T cells were transferred with antigen into irradiated F1 mice plus B cells from either the two parental strains or from F1 mice. F1 T cells activated in F1 mice gave high IgM and IgG anti-SRC responses with all three populations of B cells. By contrast, F1 T cells activated in mice of one parental strain collaborated well with B cells of this strain, but poorly with B cells of the opposite strain. Active suppression was considered an unlikely explanation for this result since (a) good responses were found with F1 B cells, and (b) addition experiments showed that the poor response with B cells of the opposite parental strain (which was equivalent to that produced by unprimed F1 T cells) could be converted to a high response by a supplemental injection of F1 T cells activated in F1 mice. The phenomenon (a) was specific for the antigen used for activation (criss-cross experiments were performed with horse erythrocytes), (b) was reflected in levels of serum hemagglutinins as well as in numbers of splenic plaque-forming cells, (c) applied also to comparable activation of (DBA/2 X C57BL/6)F1 T cells, and (d) could be prevented by activating F1 T cells in mice of one parental strain in the presence of peritoneal exudate cells of the opposite parental strain. The hypothesis was advanced that F1 T cells contain two discrete subpopulations of antigen-reactive cells, each subject to restrictions acting at two different levels: (a) during T-macrophage interactions and (b) during T-B collaboration. It was suggested that when F1 T cells are activated to antigen in a parental strain environment, radioresistant macrophages activate only one of the two subgroups of T cells, and this subgroup is able to collaborate with B cells of the strain used for activation (and with F1 B cells) but not with B cells of the opposite parental strain. The other subgroup of T cells remains in an unprimed (nonactivated) state.