From an acetone precipitate of bovine thymus a factor was isolated which stimulated the proliferation of thymocytes in vitro, without affecting growth of peripheral lymphocytes, bone marrow or liver cells. The stimulating material was purified by ultrafiltration, ion-exchange chromatography, gel filtration and semipreparative thin-layer chromatography. The activity resided in an acidic, hydrophilic low molecular weight molecule, apparently devoid of reactive amino groups. The effect was abolished by acidic hydrolysis but resisted treatment with DNase, RNase and trypsin. The effect was independent of serum and choice of culture medium. A mean increase of [3H]thymidine incorporation of 64% (range 47--101%) was obtained in thymic cells after 5 h of culture. The effect was dose-dependent and declined after 10 h. The mitotic activity increased correspondingly. A possible role of the factor in the regulation of proliferation in the thymus in vivo is suggested. The early kinetics of spontaneously proliferating thymocytes in cultures is discussed and compared to the intense growth and cell death in the thymus in vitro.