Thymus humoral factor (THF), a thymus hormone which participates in the processes leading to acquisition of immunocompetence of lymphoid cells has been isolated in our laboratory by a stepwise gel filtration through various Sephadex columns. THF so isolated appears to be a polypeptide of 3,000 mol wt which contains approximately 30 amino acid residues. Here we have tested the biological activity of THF fractions of successive degrees of purity upon lymphoid cells from both intact and neonatally thymectomized mice. The lymphoid cell populations were treated with the various THF fractions by in vitro incubation for a short time and by repeated injection in vivo. The treated cells evidenced increased ability to react in the graft-versus-host assay in vivo and in mixed lymphocyte cultures in vitro concomitantly with the rise of intracellular cAMP. On the other hand no activity whatsoever was shown by any of the control materials tested. These bioassays permitted isolation of fractions progressively more active than the original crude dialyzate of thymus extract tested. Thus the active peptide component of THF eluted from DEAE Sephadex A-25 column was estimated to be 2 X 10(4)-fold more active than the crude dialyzate of thymus extract which served as a starting material.