Vitreous humour (VH) is a useful medium for postmortem analysis. During the supravital period, anaerobic glycolysis that occurs results in the increased lactate concentration (L) in cytosol. We analysed the concentration of lactic acid in 567 samples of VH from 63 autopsy cases by a repetitive withdrawal of VH from the same corpse, while the outside temperature remained constant. The samples were taken from the same eye in intervals of three hours for the period of 24 hours. The subjects were divided into two groups: group 1 included individuals who died during winter and were kept for analysis at 40C, while group 2 consisted of those who died during spring and autumn (continental climate) and were stored at 200C. Only the cases of a sudden traumatic death of previously healthy individuals were included in the study. Statistically significant correlation of L against postmortem interval (PMI) was observed in the group 1 experiment (r = 0.675; P < 0.01), but the effect of hypothermia made the estimation of PMI practically impossible. A much stronger correlation of L against PMI was observed in group 2 (r = 0.866, P < 0.01); a functional relationship between PMI and L were demonstrated using the formula PMI = 1.696 x L - 10.562, which enabled more accurate prediction of the PMI.