Two hundred and twenty-three vitreous humor specimens, which were obtained from a medical examiner's office, were found to be opiate positive (cutoff, 50 ng/mL) by fluorescence polarization immunoassay. All samples were analyzed for their free codeine, morphine, and 6-acetylmorphine contents by a gas chromatography-mass spectrometry protocol. 6-Acetylmorphine was found (cutoff, 10 ng/mL) in 41 specimens in the concentration range of 10-125 ng/mL. Twenty specimens had a free codeine-free morphine concentration ratio > or = 1. Eighty-five samples that were found to contain 50 ng/mL free morphine were further analyzed for their total codeine and total morphine contents. Total codeine-total morphine concentration ratios in 8 (of the 85 samples) were > or = 1, whereas this ratio in the others (only those with a codeine concentration high than 15 ng/mL were included) was significantly lower than 1. The codeine-morphine concentration ratio in vitreous humor appears to resemble that reported for blood and urine and can be used as the basis for differentiating between codeine- and morphine- (heroin-) induced fatalities.