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A radioimmunoassay (RIA) capable of determining blood ACTH levels in salmonid fishes was developed and validated. The RIA used an antibody raised against mammalian ACTH, iodinated human ACTH as tracer, and human 1-39 ACTH as standard. Incubation of the standard or unknown with antibody for 3 days before addition of as little high-specific activity tracer as practicable (1500 cpm; equivalent to 5 pg ACTH) produced a very sensitive RIA; the operating range was 5 to 200 pg ACTH/ml. Extracts of both pars distalis and neurointermediate lobe of the pituitary glands from a range of salmonid species diluted parallel to the ACTH standard in the RIA. There was always considerably more ACTH-immunoreactivity (ACTH-IR) in the pars distalis extracts than in the neurointermediate lobe. Generally plasmas also diluted parallel to the ACTH standard, with the exception only of the plasma from sexually mature female salmonids, which diluted very non-parallel to the standard, leading to unrealistically low estimates of the ACTH-IR level. The use of heparin as an anticoagulant during collection of samples caused problems when these plasmas were immunoassayed; instead EDTA was found to be a suitable anticoagulant. When the ACTH-IR was extracted from a pool of plasma obtained from acutely stressed salmon and chromatographed on a column of BioGel P6, followed by subsequent ACTH RIA of the fractions, only a single sharp peak of ACTH-IR was detected, which eluted in the position of authentic 1-39 ACTH. The plasma ACTH-IR level in unstressed fish was low, and near the detection limit of the RIA. An acute stress, produced by crowding and confinement for 30 min, increased ACTH-IR approximately 10-fold, and plasma cortisol levels 50-fold, but the plasma alpha-MSH level was not affected. Dexamethasone-treated fish did not respond to this stressor with any increase in either ACTH or cortisol levels.