Vapor pressure osmometry (VPO) was used to measure the number-average molecular weights (M n ) of natural organic matter (NOM) that was isolated using reverse osmosis, hydrophobic acids (HPOA) that were isolated by adsorption on XAD-8 resin, and transphilic acids (TPIA) that were isolated by adsorption on XAD-4 resin. All samples were isolated from the headwaters of the Suwannee River in southeastern Georgia, USA, in May 2012. NOM, HPOA, and TPIA samples had M n of 634 -11, 583 -8, and 498 -7 g/mol, respectively. Novel methodology was introduced wherein VPO measurements were made at room temperature, and M n values were rendered from a robust analysis of data that considered not only the well-known effect of hydronium ion but also the contributions of inorganic solutes present in the samples. The method was validated by results on a known sample (benzenehexacarboxylic acid), whose molecular weight was determined accurately to within 0.5% of the true value. The VPO-determined M n are compared to those from noncolligative methods to highlight uncertainty regarding a fundamental parameter of matter found in nearly all terrestrial waters.