Phospholipidosis, or intracellular accumulation of phospholipids, is caused by specific classes of xenobiotics. This phenomenon represents a challenge for risk assessment that could benefit from the use of biomarkers in the clinical development of new drug candidates. Flow cytometry, coupled with the lipophilic fluoroprobe Nile red, was correlated to histopathology, electron microscopy and inorganic phosphorus detection to validate the utility of this method for monitoring phospholipidosis in peripheral blood leukocytes. Replicate studies with model test compounds were conducted in which F344 rats were given 4 or 7 doses of either maprotiline hydrochloride, imipramine hydrochloride, tilorone dihydrochloride, amikacin hydrate or vehicle control. Transmission electron and light microscopy were used to examine peripheral blood smears and tissue samples for the presence of cytoplasmic vacuoles. Unstained and Nile red stained lysed peripheral blood samples were acquired for analysis using a FACScan flow cytometer. Inorganic phosphorus concentration in the liver was determined from extracted phospholipids and compared with flow cytometry and histological data. It was demonstrated that flow cytometric analysis of Nile red stained lysed whole blood can be used to detect drug-induced phospholipid accumulation in circulating peripheral leukocytes. Furthermore, clinically detectable leukocyte phospholipidosis may be a useful surrogate for coincident or premonitory detection of target organ phospholipidosis. Copyright © 2005 John Wiley & Sons, Ltd.