The technique of tear film osmolality determination using a nanoliter freezing point depression type osmometer was studied to determine what factors affect the accuracy of the technique. Reflex tearing during microcapillary sampling of the tears caused a 5% decrease in tear film osmolality. Evaporation effects of sampling, retrieval and osmolality determination of samples resulted in an increase of 0.2% for one ul volumes and 1.2% and 1.3% for 0.50 and 0.25 ul volumes of samples, respectively. Evaporation of the sample during storage between layers of immersion oil resulted in increasing osmolalities of 0.5, 0.2 and 0.2%/day for sample volumes of 0.25, 0.50 and 1.0 ul respectively over 14 days. Samples (1.0 ul) stored at 4®C showed an increase in osmolality of 1.25 mos/kg/day. These results suggest that reflex tearing is one major source of variation in tear film osmolality determiniations and that avoidance of reflex tearing is critical to the accuracy of the technique.