A single injection of immunodeficient old (15-24 month old) BDF1 mice with 1-100 micrograms of synthetic thymosin alpha 1 (T alpha 1), a 28-amino acid residue peptide, shortly before horse erythrocyte (HRBC)-priming, enhances the helper activity of their spleen cells. Helper activity of spleen cells from uninjected or T alpha 1-injected HRBC-primed old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from unprimed young (3-month-old) mice and the conjugate 2,4,6-trinitrophenyl (TNP)-HRBC. As evaluated from the in vitro anti-TNP antibody response, spleen cells exhibit higher helper activity when derived from T alpha 1-injected, as compared to uninjected, HRBC-primed old mice. No effect is observed when T alpha 1 is injected in young mice before HRBC-priming. Old mice were also injected with the N14 (N-terminal amino acid residues 1-14) or the C14 (C-terminal amino acid residues 15-28) synthetic fragments of the T alpha 1 molecule. Injection of T alpha 1 or the N14 fragment restores the helper activity of old mice to levels comparable to that displayed by helper cells from young mice. Conversely, injection of the C14 fragment in old mice induces only a negligible increase in helper activity of their spleen cells. T cells from HRBC-primed mice were separated by nylon wool filtration into Th1 (non-adherent) and Th2 (adherent) T cells and these cell subpopulations were assayed for helper activity. Helper activity of Th1 and Th2 cells is found to be impaired by aging when they are tested either separately or upon their recombination.(ABSTRACT TRUNCATED AT 250 WORDS)