The in vitro production of gamma-hydroxybutyrate (GHB) in antemortem urine samples was demonstrated over an eight-month period. Positive chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS) was used to detect trace amounts of GHB produced in vitro under certain storage conditions. Freshly prepared drug-free human urine was stored at 21, 4, and -20 degrees C in the presence of preservative. Although artifactual production of GHB occurred more rapidly at elevated temperatures, the presence of an antimicrobial agent (sodium azide) in the drug-free urine control did not impede GHB production. The preliminary data suggest that although in vitro production was demonstrated, the elevations in concentration were nominal and less than 5 mg/L for all conditions tested over the 244-day period. These preliminary data suggest that urine samples should be preserved and stored at -20 degrees C to minimize artifactual GHB production. Most importantly, conditions of storage and preservative should also be taken into consideration when interpreting GHB results that are close to the administrative cutoff. In order to establish the distribution of GHB concentrations in routine forensic case samples, a series of 100 antemortem urine samples, in which GHB was not suspected, was analyzed. Samples were preserved with sodium fluoride (1%) and had been stored for up to one year at room temperature. Although concentrations as high as 7 mg/L were measured in some samples, the mean and median concentrations were 1.8 mg/L and 1.6 mg/L, respectively. Even following storage at room temperature for an extended period, more than 95% of the urine samples contained less than 5 mg/L GHB and 100% contained less than 10 mg/L. An administrative cutoff of 10 mg/L in antemortem urine was used for routine antemortem casework.