This paper describes the production and evaluation of an antiserum with high affinity (Ka = 1.9 × 1011 l/mol) and specifity to 8-arginine-vasopressin (AVP). The antiserum binds only to the intact and unchanged ring and tail of the AVP-molecule. AVP was labelled with 125I by a modification of the chloramin T method and purified by gel-filtration. We describe the development and validation of a radioimmunoassay for AVP in human plasma and urine, using only one extraction method (octa-decasilyl-treated silica microcolumns) for both biological fluids. The overall sensitivity of the assay method is 0.3 pg/ml plasma and 0.6 pg/ml urine. Mean (± sd) plasma AVP-concentration in normally hydrated females was 1.2 ± 0.6 pg/ml (median 1.2 pg/ml) and 1.7 ± 0.7 pg/ml (median 1.8 pg/ml) in males. Mean urinary AVP excretion was 73 ± 43 ng/day with ad libitum water intake and normal activity. Sex differences were not statistically significant. We also assessed the response of plasma AVP and urinary AVP-excretion to waterload and dehydration.