Malachite green is a known teratogen and therefore its use is limited to nonfood fish under an Investigational New Animal Drug permit (INAD), number 2573. Although a charcoal adsorption column was developed to remove malachite green from hatchery water, INAD compliance requires that the malachite green residue concentrations in any effluent from hatcheries using the chemical be quantified. Therefore, we developed a method for the analysis of malachite green residues in water. Enrichment of the residues of malachite green in water on a diol column followed by High Performance Liquid Chromatographic (HPLC) analysis gives a minimum sensitivity of less than 10 ppb for the chemical. When combined with post-column oxidation using a lead oxide post-column reactor, the procedure can be used for the simultaneous analysis of malachite green in its leuco form, a decomposition product of the dye, as well as its chromatic form. Recovery of the leuco form is pH dependent and water samples should be adjusted to pH 6 to optimize recovery of this form. Water samples spiked with malachite green were concentrated on a diol column followed by elution with 0.05 M p-toluene sulfonic acid in methanol. The methanol elutes were analyzed by HPLC. Pond water samplesmore » spiked with malachite green and leuco malachite green yielded average recoveries of 95.4% for malachite green and 57.3% for leuco malachite green. Tap water samples spiked with the carbinol form of malachite green gave average recoveries of 98.6%. The method is very sensitive and is capable of detecting malachite green residues in water at less than 10 ppb. Fish culturists, who cannot find an effective replacement for malachite green, can utilize the method to ensure that their effluents comply with INAD regulations. 13 refs., 2 figs., 7 tabs.« less