A simple, rapid and sensitive normal-phase high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of a novel hypolipidemic agent in human feces, plasma and urine. This experimental drug candidate is structurally related to rifamycin. The compound and internal standard were isolated from biological matrices by a one step liquid-liquid extraction. Separations were achieved on a mu Porasil silica gel column. Recovery and reproducibility assessments indicated good accuracy and precision. The overall mean relative recoveries were 93.3% from feces (0.2-20 micrograms/mg), 95.1% from plasma (20-500 ng/ml) and 97.5% from urine (20-500 ng/ml), with coefficients of variation ranging from 0.7 to 10.0% for feces, 3.0 to 12.7% for plasma and 2.3 to 10.6% for urine. The limits of quantification were 0.2 micrograms/mg for feces and 20 ng/ml for plasma and urine. The method has sufficient sensitivity to support clinical trials, and was utilized to measure concentrations of the compound in fecal, plasma and urine samples from healthy male volunteers who had received a single 800-mg oral dose.