In the gravimetric method for estimation of water content in brain tissue, four factors that may affect specific gravity (SG) measurements are described and evaluated. These factors are: (1) the purity of kerosene (K) and monobromobenzene (MBB), (2) duration of interaction of MBB with tissue and/or standard solutions, (3) the size of tissue samples, and (4) the temperature. A water-soluble contaminant in K, which can be effectively extracted by water treatment, may significantly decrease SO values. Conversely, in MBB, the SG of tissue samples tends to increase in proportion with time. The SG of the tissue samples appears to be influenced by their size, the smaller pieces registering higher SG values. Also, the temperature of the column affects the SG, with higher temperatures resulting in higher SG values; a difference of 5°C produces a significant difference in SG (p<0.05). A standardized procedure providing highly reproducible SG values is presented in which the size of the tissue samples, the recording time, and the temperature of the column consisting of water-treated K and MBB are kept strictly constant.