Thymosin fraction 5 (TF5) is a partially purified preparation of bovine thymus that affects the differentiation and function of T-cells in vitro. Interleukin-6 (IL-6) is a pleiotropic cytokine that induces terminal maturation of B-cells and T-cell activation and differentiation. Although TF5 had previously been shown to stimulate the production of a number of lymphokines, its effects on IL-6 were not known. In this study we determined the effect of TF5 on IL-6 production from rat spleen cells in vitro. TF5 (100 micrograms/ml) stimulated IL-6 production from splenocytes (0.75-3.0 x 10(5) cells/well) in the presence of 0.008-0.2 micrograms/well of the T-cell mitogen concanavalin-A (con-A) by 10-20 fold during a 72 h incubation period. Dose-response studies demonstrated that 10 micrograms/ml of TF5 was the lowest concentration capable of enhancing IL-6 production. The ability of TF5 to stimulate IL-6 production in the presence of con-A could be demonstrated within 24 h of incubation; longer incubation periods (48-72 h) correlated with further enhancements of IL-6 production. Partial purification of the IL-6-inducing activity from TF5 resulted in three subfractions possessing activity in the presence of con-A (MB2, MB3, MB7) and one in the absence of con-A (MB2). The previously characterized thymosin peptides T alpha 1 and T beta 4 had no effect on IL-6 production in the absence or presence of mitogen. This study reports a new biological activity for TF5 and suggests that a novel constituent of TF5 may enhance the production of IL-6 from spleen cells.