A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new isoquinolinedione derivative, 7-anilino-5,8-isoquinolinedione (IQO4), in rat plasma, urine, blood and tissue homogenates using diazepam as an internal standard. A 2 volume of acetonitrile was added to deproteinize the biological sample. A 50 microl aliquot of the supernatant was injected onto a C(18) reversed-phase column. The mobile phase, 0.05 M acetate buffer (pH 3):acetonitrile:methanol (40:40:20, v/v/v), was run at a flow rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 298 nm. The retention times for IQO4 and the internal standard were approximately 5 and 7 min, respectively. The detection limits of IQO4 in rat plasma, urine and tissue homogenates (including blood) were 0.05, 0.1 and 0.1 microg/ml, respectively. The coefficients of variation of the assay were below 9.4% for rat plasma, urine and tissue homogenates. No interferences from endogenous substances were found.