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Upon contact with water, under a variety of conditions, C60 spontaneously forms a stable aggregate with nanoscale dimensions (d = 25−500 nm), termed here “nano-C60”. The color, hydrophobicity, and reactivity of individual C60 are substantially altered in this aggregate form. Herein, we provide conclusive lines of evidence demonstrating that in solution these aggregates are crystalline in order and remain as underivatized C60 throughout the formation/stabilization process that can later be chemically reversed. Particle size can be affected by formation parameters such as rates and the pH of the water addition. Once formed, nano-C60 remains stable in solution at or below ionic strengths of 0.05 I for months. In addition to demonstrating aggregate formation and stability over a wide range of conditions, results suggest that prokaryotic exposure to nano-C60 at relatively low concentrations is inhibitory, indicated by lack of growth (≥0.4 ppm) and decreased aerobic respiration rates (4 ppm). This work demonstrates the fact that the environmental fate, distribution, and biological risk associated with this important class of engineered nanomaterials will require a model that addresses not only the properties of bulk C60 but also that of the aggregate form generated in aqueous media.