Guanylin is a peptide isolated from rat intestine that stimulates intestinal guanylate cyclase. We describe here the purification of circulating guanylin from human hemofiltrate. By N‐terminal protein sequence analysis 47 amino acids were determined. This sequence corresponds to the positions 22 to 68 of the prohormone deduced from the cDNA sequence of human proguanylin. Mass spectral analysis of the circulating peptide showed the molecular weight to be 10,336 Da, which corresponds to the mass calculated from position 22 to the C‐terminus of the peptide predicted from the cDNA sequence. Circulating guanylin markedly increased the cyclic GMP content of T84 cells. Our data show that the hormonal form of guanylin is circulating as a 10.3‐kDa peptide in human blood.