Mononuclear leukocytes from 25 children (16 with normal pituitary ACTH production and 9 with ACTH deficiency) were examined for in vivo ACTH production by immunofluorescence with antiserum to ACTH-(1-13) amide. The protocol included 3 study periods: control, after administration of insulin, and after administration of typhoid vaccine (an interferon-a inducer). Plasma cortisol and mononuclear leukocyte ACTH immunofluorescence were measured before (0900 h) and 2, 4, 6, 8, and 10 h after treatment on each of the 3 study days. In vitro studies with human leukocytes from normal subjects incubated with ACTH, insulin, or typhoid vaccine were also performed. Patients with normal pituitary ACTH production had an increase in the number of ACTH immunofluores-cence-positive cells 1 h after insulin administration [25 ± 5% (± SEM) to 44 ± 6% P < 0.05], and no change after typhoid administration. ACTH-deficient patients had no change after insulin administration and a significant rise 6 h after typhoid vaccine treatment (24 ± 12% to 50 ± 6%; P < 0.05). The number of ACTH immunofluorescence-positive cells did not increase when mononuclear leukocytes were incubated in vitro with ACTH or insulin (with or without glucose deprivation). However, typhoid antigen enhanced this response from 8% to 55%. These data suggest that the number of human mononuclear leukocytes containing immunoreactive ACTH is increased by at least 2 stimuli: 1) a central factor(s), such as CRH, accounting for the in vivo rise 1 h after insulin administration in patients with an intact hypothalamic-pituitary axis, and 2) an interferon inducer (e.g. typhoid antigen), accounting for the typhoid antigen-induced rise in the number of ACTH-positive cells in vivo in ACTH-deficient patients and in vitro. © 1987 by The Endocrine Society.