The objective of the present study was to determine whether or not thymosin and conditioned medium from human thymus epithelial cultures (HTECM) contain similar fractions, capable of inducing T-cell differentiation. Therefore we tested the ability of rabbit antisera to different thymosin fractions (thymosin fractions 5, 6 and alpha 1) of both bovine and human origin to block the stimulatory effect of HTECM on Con A and PHA response of mouse thymocytes. We also looked for reactivity of the antisera toward tissues of man, mouse and calf, and to tissue cultures of man and mouse. Anti-thymosin fraction 5 and 6, but not anti-thymosin-alpha 1, were found to inhibit the stimulatory effect of HTECM on both Con A and PHA responses. This cannot be attributed to cytostatic or cytotoxic effects of the antisera on thymocytes. No effect was seen when using antiserum to kidney fraction 5 or normal rabbit serum. In both tissue sections and tissue cultures of the different species tested, anti-thymosin reacts only with thymus epithelial cells. The reactivity is blocked by neutralizing the antisera with thymosin fraction 5 but not by kidney fraction 5. The reactivity is also strongly diminished by neutralizing the antisera with HTECM but not with supernatants of conditioned media from non-thymic tissues. The observations are highly suggestive for the presence of similar fractions in HTECM and thymosin, and that both are secreted by thymus epithelial cells.