A differential pulse polarographic assay for plasma vitamin K3 (menadione) has been developed. Details of the assay are (i) lipid-soluble material is extracted from plasma into ether by the method of Bjornsson et al. [(1978) Thromb. Haemostas. 2, 466-473]; (ii) ether is evaporated under nitrogen and the residue is dissolved in the supporting electrolyte, methanol: 0.2 M borate buffer (9:1), pH 6.8; (iii) current height is measured at -0.32 V vs SCE on the differential pulse polarogram. The lower sensitivity limit of this technique after addition of standard vitamin K3 to plasma is 0.3 microM; the calibration curve is linear from 0.6 through 10 microM. Two patients treated with a single dose of menadiol sodium diphosphate, 20 mg/M2 i.m., achieved measurable plasma vitamin K3 levels at 0.5 to 1.0 h ranging between 0.5 (0.08 micrograms/ml) and 2 microM (0.3 micrograms/ml).