Two-dimensional thermal field-flow fractionation (2D-ThFFF) is a new instrumental technique devised for continuous fractionation of soluble macromolecules and particles. The sample mixture is introduced into a disc-shaped channel and the separated sample components are collected continuously from the channel outlets. The method is based on a two-dimensional fractionation mechanism with radial and tangential flow components in the channel. The effects of flow components and thermal gradient on the fractionation were studied in the separation of polystyrene samples of different molecular masses using cyclohexane or a binary solvent consisting of 25% ethylbenzene and 75% cyclohexane as carrier. The continuous separation of polystyrene samples was improved with increasing thermal gradient and with the use of slow radial and tangential flow rates. The technique can be applied to preparative continuous separation of macromolecules.