Compositional grading of spherules in the ~3.24 Ga spherule bed S3, Barberton greenstone belt, South Africa, provides constraints on the evolution of the impact-generated rock vapor plume within which the spherules formed. Two fall-deposited sections of S3, 40 km apart, are both about 30 cm thick and composed almost entirely of spherules. These spherules are diverse in size, composition and texture. Although they have been chemically altered, primary textures and some mineralogy is preserved. Both sections show a similar compositional grading, which includes five stratigraphic zones, each composed of a distinctive assemblage of spherule types. Petrographic and geochemical data suggest that this stratigraphic layering is the same in both sections. This regionally consistent grading suggests large-scale compositional and thermal layering in the impact plume. REE data indicate that the basal part of S3 in these sections contains the most refractory material and that the upper part contains the most basaltic components. Overall, the beds show a temporal evolution consistent with the condensation sequence predicted for a fractionation-modified mixing of chondritic and basaltic materials.