Aim: While nanoparticles (NPs) have been shown to form spontaneously in body fluids such as serum, the possible implications of these NPs for cell cultures that use supporting media containing serum remain unclear. To understand the de novo formation of NPs, we delineated their growth characteristics, chemical composition and interaction with cells in culture. Materials & Methods: Serum-derived particles were analyzed using a combination of dynamic light scattering, turbidity measurements, spectroscopic techniques and optical/electron microscopies. Results: NPs were found in serum and in serum-containing medium and they increased in size and number during incubation. The mineral particles, consisting mainly of calcium carbonate phosphate bound to organics such as proteins, underwent an amorphous-to-crystalline transformation with time. Serum-derived particles were internalized by the cells tested, eventually reaching lysosomal compartments. Conclusion: The spontaneous formation of serum-derived NPs and their internalization by cells may have overlooked effects on cultured cells in vitro as well as potential pathophysiological consequences in vivo.