Postmortem heart blood, peripheral blood, vitreous humor, urine, and bile specimens from 26 autopsy cases were analyzed for the presence of gamma-hydroxybutyric acid (GHB) and gamma-methyl gamma-hydroxybutyric acid (4-Me-GHB) after long-term freezer storage. Cases were selected for which exogenous GHB, gamma-butyrolactone (GBL), gamma valerolactone (GVL), or 1,4-butanediol use was not suspected. One documented positive GHB case subjected to the same storage conditions was also evaluated for comparison. Specimens did not contain any preservatives or additives except heart blood, which contained sodium fluoride (2% w/v). The results of the analysis for GHB in vitreous humor (n = 26) demonstrated, with one exception, concentrations below the limit of detection for the method (5 mg/L). In the exception case, the value was determined to be 7 mg/L. Documented cases of GHB positive fatalities showed vitreous humor concentrations (n = 6) that exceeded this range by a factor of 12 or more. There was no apparent relationship between storage times and GHB concentrations. The data developed in this study demonstrate a postmortem endogenous range for GHB in vitreous humor that is less than or equal to 7 mg/L. Studies of the stored GHB-positive case demonstrated no significant change in concentration over the time period studied. None of the specimens analyzed in this study contained detectable amounts of 4-Me-GHB. This would support the contention that when 4-Me-GHB is detected, it is most likely due to the exogenous consumption of GVL.