The electrophoretic mobility (EPM) and the ability to synthesize DNA in vitro either spontaneously or in response to concanavalin A (Con A) and phytohemagglutinin (PHA) were investigated on thymocytes from adult female Swiss/B mice which had received intraperitoneally, 2 days previously, various doses (10–750 mg/kg body weight) of hydrocortisone acetate (OHC). A first group of thymocytes, highly sensitive to OHC, rapidly decreased in proportion following administration of increasing doses of OHC (10–125 mg/kg body weight) and included the totality of low-EPM thymocytes (EPM < 1.0 μm sec––1 V––1 cm) together with about 70% of the high-EPM thymocytes encountered in the normal thymus. The second group of thymocytes (4% of the initial cellularity), which resisted to OHC at 125 mg/ kg body weight and was only reduced by half with doses of OHC 6-fold larger, was composed essentially of high-EPM cells. The mean EPM of both groups tended to increase with increasing doses of OHC which suggests that among these two groups thymocytes are all the more OHC-sensitive as they possess a low surface charge. Spontaneous DNA synthesis, measurable on the first 24 h of in vitro cultivation, markedly dropped after treatment with OHC at doses higher than or equal to 62 mg/kg body weight. This loss of early spontaneous proliferation coincided with the disappearance of medium-EPM thymocytes. By contrast, proliferative response of thymocytes to mitogens was enhanced by OHC pretreatment of donors and roughly paralleled the enrichment in high-EPM cells. However, administration of the largest amounts of OHC diminished this mitogen responsiveness, although PHA reactivity was less affected than Con A reactivity.