A recent report measured a decrease in plasma ACTH concentration by immunoradiometric assay (IRMA) during infusion of ACTH-(1-24) in humans. It was concluded that this decrease in ACTH concentration was due to short loop ACTH autoregulation. The present study demonstrates that the decrease in ACTH concentration measured by IRMA was due to an artifact of the IRMA. We injected 250 micrograms ACTH-(1-24), iv, into five normal male volunteers after overnight 2.5-g metyrapone administration. The ACTH concentration measured by IRMA decreased from 59.6 +/- 9.7 pmol/L before to 4.8 +/- 2.0 pmol/L 1 min after ACTH-(1-24) injection. The ACTH concentration measured by IRMA increased thereafter in a mirror image of the decline in ACTH-(1-24) measured by RIA. Addition of ACTH-(1-24) to plasma in vitro resulted in a decrease in the ACTH concentration measured by IRMA which was of similar magnitude to that observed in vivo. ACTH-(1-24) infusion in vivo or addition to ACTH-(1-39)-containing plasma in vitro decreased ACTH-(1-39) measured by IRMA by binding to N- but not C-terminal antibody without forming a detectable sandwich complex. We conclude that although ACTH short loop feedback may exist, it cannot be detected after ACTH-(1-24) injection with the use of a two-site IRMA.