4-Nonylphenol glucuronide (NP-G) in human urine samples was analyzed using stir bar sorptive extraction (SBSE) with in situ de-conjugation by beta-glucuronidase and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Distilled water (1 ml), 1.0 M ammonium acetate solution (100 microl) and beta-glucuronidase (10,000 units ml(-1), 10 microl) were added to human urine sample (1 ml), and extraction was commenced for 90 min at 37 degrees C while stirring at 250 rpm with a stir bar coated with a 500-microm-thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was subjected to TD-GC-MS in the selected ion monitoring (SIM) mode. The calibration curve was made by SBSE method using 4-nonylphenol (NP) as the standard solution. The method showed good linearity and the correlation coefficients were 0.999 over the concentration range of 5-500 nM. Moreover, to optimize the conditions for SBSE with in situ de-conjugation and the recovery test, NP-G was synthesized by a biochemical technique in our laboratory. The limits of detection (S/N = 3) and quantitation (S/N > 10) for NP were 0.2 ng ml(-1) (1.0 nM) and 1.1 ng ml(-1) (5.0 nM), respectively. The average recoveries in the human urine samples (n = 6) spiked with NP-G at levels of 20 and 100 nM were 104.1 (R.S.D. 7.1%) and 100.6% (R.S.D. 9.2%), respectively, with correction using the added internal standard, 4-(1-methyl) octylphenol-d(5). The method enabled the precise determination of the standard and was applicable to the detection of trace amounts of NP-G in human urine samples.