Vitreous humor (VH) can provide valuable forensic evidence regarding the time and manner of death because it is known to be a more stable postmortem (PM) than either blood or CSF. Additionally, VH analysis can reflect antemortem plasma concentrations of sodium (Na), chloride (Cl), and urea nitrogen (UN), making VH an ideal postmortem specimen for analysis of chemical analytes. The goals of the current study were threefold: (1) to evaluate the stability and reproducibility of refrigerated VH specimens for the measurement of Na, Cl, and potassium (K) over a 6- to 12-month period; (2) to compare levels of beta-hydroxybutyrate, acetone, and acetonacetate concentrations; and (3) to compare calculated versus measured osmolality concentrations in a subset of frozen VH specimens. Between 2001 and 2002, VH specimens were collected from both eyes at autopsy from 126 medical examiner cases. Statistical analyses revealed that small but significant increases in VH electrolyte concentrations were observed in specimens refrigerated 6 to 12 months. Within-day precision for all electrolyte measurements was observed to be excellent and no differences were observed between eyes. The frozen specimens showed significantly higher measured osmolality compared with calculated osmolality; a 1 percent within-day precision was observed and there was no significant between eye variation in glucose and urea nitrogen. In 20 of the 24 cases selected for possible ketoacidosis, measurements of beta-hydroxybutyrate concentrations seemed to be a promising biomarker for confirmation of suspected ketosis. The findings thus indicate that specimens should be stored frozen and that the Hitachi system for VH analyte determinations is a reliable methodology. Tables, references