We developed a one-step two-site immunoradiometric assay (IRMA) using two kinds of monoclonal antibodies, which enables us to directly measure the entire molecules of adrenomedullin (AM) (the sum of mature-type AM (abbreviated, m-AM) amidated at the C-terminus and Gly-extended non-amidated AM) in human plasma using a small amount of sample (100 microl) without prior extraction. The detection limit of this assay was 0.5 pmol/l for a 100-microl sample. Intra- and inter-assay precisions were 3.4-7.3% and 5.8-7.6%, respectively. The dilution curves of plasma samples showed good linearity and analytical recovery was 89-118%. The mean total AM in plasma of healthy subjects was 9.00+/-2.13 pmol/l, whereas m-AM was 1.05+/-0.24 pmol/l. This method, together with our previously reported simplified method to specifically measure m-AM (Ohta et al., Clin Chem 1999;45:244-251), allows facile estimation of the plasma concentration of AM-Gly by subtracting m-AM from the total AM measured by the procedure described in this paper. We were able to show that the concentration of total AM in patients with sepsis was markedly higher than that in the healthy controls and that the ratios of m-AM/total AM were significantly different between the controls and patients.