The mechanisms involved in the activation of the hypothalamus-pituitary-adrenal axis after administration of Mycoplαsma fermentans were examined. Male rats were injected intracerebroventricularly (i.c.v.) with heat-inactivated M. fermentans (6 µg protein/10 µl/rat) or vehicle and were killed 2 h later. M. fermentans caused a significant depletion of corticotropin releasing hormone (CRH-41) content in the median eminence (ME), and elevation of serum ACTH and corticosterone (CS) levels, compared to control levels. Pretreatment with dexamethasone (DEX, 40 µg/kg) markedly inhibited M. fermentans-induced depletion of ME CRH-41 and the increase in serum ACTH and CS. Injection of the type II corticosteroid receptor antagonist RU-38486, but not the type 1 antagonist RU-28318, enhanced the adrenocortical response and completely abolished the inhibitory effect of DEX following M. fermentans. Injection of the catecholamine neurotoxin 6-hydroxydopamine into the ventral noradrenergic bundle, which significantly depleted hypothalamic nor-epinephrine content, or i.c.v. injection of the specific α<sub>1</sub>-adrenergic receptor antagonist prazosin failed to affect the adrenocortical response to M. fermentans. In contrast, these agents markedly inhibited the adrenocortical response following i.c.v. injection of interleukin-1. I.c.v. administration of M. fermentans caused a significant elevation of hypothalamic levels of tumor necrosis factor-α (TNFα), determined by both bioassay and immunoassay. In rats treated with pentoxifylline, an inhibitor of TNFα synthesis, the adrenocortical response to M. fermentans was markedly inhibited. These findings suggest that: (1) M. fermentans-inducedactivation of the pituitary-adrenal axis, and the inhibitory effect of DEX on this response, are mediated by a reduction of CRH-41 release from the ME. (2) The feedback exerted by glucocorticoids is mediated by type II corticosteroid receptors. (3) In contrast to the adrenocortical response to interleukin-1β, the central noradrenergic system does not have an important role in mediating the adrenocortical response to M. fermentans. (4) Hypothalamic TNFα production is probably involved in mediating the adrenocortical activation following M. fermentans.