Serum thymic factor (FTS) and its 33 analogs were compared with regard to the ability to induce Thy-1 antigen on mouse pre-T cells. The pre-T cells were prepared from spleen cells of athymic nu/nu mice by passing them through a nylon wool column, and the induction of Thy-1 antigen was analysed using a fluorescence-activated cell sorter. Thy-1 negative cells were converted into Thy-1 positive cells by FTS and some of its analogs. Deletion of pGlu at the amino terminus of FTS did not alter the activity. Des ( pGlu1 , Ala2)-FTS and des ( pGlu1 , Ala2, Lys3 )-FTS were inactive and deletion of the carboxyl terminal residue, Asn, also resulted in the loss of activity. Substitution or modification of pGlu1 and Asn9 with an amino acid, an acyl group or an amine caused a marked reduction of the activity. The residue 3, Lys, could be substituted without any loss of the activity by Arg, but not by Leu. These results show that the octapeptide moiety, Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn, is the minimum essential part of the FTS molecule for the expression of activity and they suggest that the positive charge at the residue 3 is also important for its activity.