An HPLC method was developed for the determination of thymoxamine metabolites deacetylthymoxamine (M1) and N-monomethyldeacetylthymoxamine (M2) in blood plasma. The samples were hydrolyzed with HCl to produce M1 and M2 from their sulfate and glucuronide conjugates. The samples were extracted with CH2Cl2, the organic phase was reextracted with aqueous HCl, and the new aqueous phase was reextracted with cyclohexane. The reconstituted residues of the cyclohexane phases were analyzed on a Spherisorb RP-18 column using acetonitrile-water-5.5% heptanesulfonic acid (40:54:6) as a mobile phase, 4-[2-(ethylmethylamino)ethoxy]-2-methyl-5-(1-methylethyl)phenol as an internal standard, and fluorimetric detection. The method gives linear responses in the range 5-2000 ng M1 or M2/mL and its detection limit for M1 and M2 is 5 ng/mL. The method requires only 1 mL of plasma, in contrast to most other methods which require at least 2 mL of plasma.