A size separation method was developed for aqueous C60 fullerene aggregates (aqu/C60) using asymmetric flow field flow fractionation (AF4) coupled to a dynamic light scattering detector in flow through mode. Surfactants, which are commonly used in AF4, were avoided as they may alter suspension characteristics. Aqu/C60 aggregates generated by sonication in deionized water ranged in size from 80 to 260 nm in hydrodynamic diameter (Dh) as determined by DLS in flow through mode, which was corroborated by analysis of fractions by DLS in batch mode and by TEM. The mass of C60 in each fraction was determined by LC-APPI-MS. Only 5.2+/-6.7% of the total aqu/C60 mass had Dh less than 80 nm, while 58+/-32% of the total aqu/C60 mass had Dh between 80 and 150 nm and 14+/-9.2% of the total aqu/C60 were between 150 and 260 nm in Dh. With the optimal fractionation parameters, 77+/-5.8% of the aqu/C60 mass eluted from the AF4 channel, indicating deposition on the AF4 membrane had occurred during fractionation; use of alternative membranes did not reduce deposition. Channel flow splitting increased detector response although channel split ratios greater than 80% of the channel flow led to decreased detector response. This is the first report on the use of AF4 for fractionating a colloidal suspension of aqu/C60.