Abstract A new method for the determination of ochratoxin A (OTA) in human urine samples has been developed using solid-phase microextraction (SPME) interfaced with liquid chromatography-fluorescence detection (LC-FD). This method is simpler and cheaper compared to the most widely adopted clean-up procedures for OTA extraction from urine (usually based on immunoaffinity columns). Briefly, urine samples, diluted 1:5 with phosphate buffer (10 mM, pH 3), were partitioned against chloroform and the aqueous phase extracted by a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber. The fiber was then “statically” desorbed, through a SPME interface, into a LC system operating in isocratic conditions. The linear range investigated in urine was 0.01–1 ng/ml. Within-day R.S.D.% in urine spiked at 0.1 and 1 ng/ml were 3.9 and 1.9, respectively, whereas the between-days R.S.D.% were 5.5 and 3.0, respectively. The limits of detection (LOD) and quantitation (LOQ) calculated at a signal-to-noise ratio of 3 and 10 (noise calculated peak to peak on a blank chromatogram at the OTA retention time) were 0.01 and 0.05 ng/ml, respectively.