Spleen and lymph node cells from normal or adrenalectomized (Adx) rats were assessed for a variety of in vitro immunological measures, including mitogen responsiveness, antibody-dependent cellular cytotoxicity (ADCC), and natural killer (NK) cell activity. Proliferation in response to mitogens was evaluated with or without 2-mercaptoethanol (2-ME) in the culture media. Compared to spleen cells from normal rats, those from Adx rats demonstrated enhanced responsiveness to all mitogens tested. 2-ME increased proliferation of spleen cells from both normal and Adx rats, and the effect was additive to that of adrenalectomy. In contrast, lymph node cells from Adx rats manifested decreased mitogenic responsiveness compared to those of control rats and, with the addition of 2-ME, this difference disappeared for one of the mitogens tested (phytohemagglutinin). Compared to unoperated rats, ADCC and NK activities were significantly decreased in both the spleen and lymph nodes of the Adx rats. Alteration of normal leukocyte circulatory and homing patterns are suggested as a likely means by which adrenalectomy produces differential effects on lymphoid cells in various organs of the immune system.