Activity of the hypothalamic-pituitary-adrenal axis is characterized by a diurnal rhythm with an AM nadir and PM peak. Splanchnic nerve transection disrupts the diurnal rhythm in plasma corticosterone, however there is a controversy as to whether the nerve-mediated effect is (1) via inhibition in the AM versus excitation in the PM, or (2) involves changes in adrenal sensitivity to ACTH. The present studies were designed to address these issues. Adult male rats were anesthetized and underwent bilateral transection of the thoracic splanchnic nerve or shamtransection. One week after surgery, rats were killed in the AM or PM with collection of nonstress plasma for measurement of corticosterone and ACTH. Plasma corticosterone was increased in the PM relative to the AM; however, plasma corticosterone in the PM was attenuated by splanchnic nerve-transection, without affecting plasma ACTH. This decrease in PM plasma corticosterone after nerve-transection was (1) associated with decreased adrenal responsivity to ACTH, (2) associated with decreased adrenal cAMP content, (3) prevented by adrenal demedullation, and (4) not affected by removal of adrenal capsaicin-sensitive afferent fibers. Repeated serial blood sampling from individual rats confirmed the excitatory effect of splanchnic innervation in the PM. These results support the hypothesis that the adrenal splanchnic innervation modulates the diurnal rhythm in plasma corticosterone by increasing adrenal responsivity to ACTH and augmenting steroidogenesis in the PM, and suggest that alterations in adrenal corticosterone secretion obscured by pulsatile secretion are more clearly revealed with repeated serial blood sampling.