Solid-phase microextraction (SPME) is a unique extraction and sampling technique, and it has been used for separation of volatile organics from water or other simple matrices. In this study, we have used SPME to separate dinitroaniline herbicides from complicated matrices of human urine and blood in order to broaden its application to biomedical analysis. The SPME conditions were optimized for water, urine and blood samples, in terms of pH, salt additives, extraction temperature, and fiber exposure time. Urine or water (1.0 ml) spiked with herbicides and 0.28 g of anhydrous sodium sulfate was preheated at 70 degrees C for 10 min, and a polydimethylsiloxane-coated fiber for SPME was exposed to the headspace at 70 degrees C for another 30 min; while spiked blood (0.5 ml) diluted with water (0.5 ml) was treated at 90 degrees C in the same way. The herbicides were extractable under these conditions, and could be determined by gas chromatography-electron capture detector (GC-ECD). The recoveries of the herbicides, measured at the concentrations of 0.50 and 1.0 ng/ml urine or water, or 6.0 and 20 ng/0.5 ml blood, ranged from 35 to 64% for different herbicides from water or urine, and from 3.2 to 7.2% from blood. The headspace SPME yielded clean extracts of dinitroaniline herbicides from urine, blood or water, which could be directly analyzed by GC-ECD without further purification. The peak areas of the extracted herbicides were proportional to their concentrations in the range 0.1-10 ng/ml in water or urine, or 1-60 ng/0.5 ml in blood. The lowest detectable concentration of the herbicides lay in 0.1 ng/ml water or urine, or in 0.5 ng/0.5 ml blood. The intra- and inter-day coefficients of variation were within 14% for most of the analytes. Although the recoveries of the herbicides were rather low, the linearity of calibration curve and the precision were good. The developed method is more sensitive and much simpler in sample preparation than previously reported ones. With the established SPME method, a dosed herbicide was successfully separated and determined in rats' blood.