Sep-Pak C18 extraction of human plasma and radioimmunoassay using antibodies which recognize atrial natriuretic peptide (99-128) and the prohormone sequences 1-30 and 31-67 resulted in mean values from 20 normal subjects of 26.2 (±9.2), 362 (±173) and 368 (±160) pg/ml, respectively. A high correlation coefficient between values obtained using antibodies recognizing prohormone sequences 1-30 and 31-67 was observed (R=0.84). Extracted plasma immunoreactivity of 1-30 and 31-67 both eluted at 46% acetonitrile. In contrast, chromatographic elution of synthetic peptides 1-30 and 31-67 was observed at 48 and 39% acetonitrile, respectively. Data suggest that the radioimmunoassay of plasma using antibodies recognizing prohormone sequences 1-30 and 31-67 may represent the measurement of a unique larger amino-terminal peptide fragment containing antigenic sites recognized by both antisera. © 1990.