Cells are crowded places, with macromolecule concentrations exceeding 100 mg/mL in eukaryotic cells. Yet in biochemical experiments seeking to reproduce intracellular conditions, controlling for solution crowding is often overlooked. Actin polymerization is a fundamental cellular process driven by nucleotide hydrolysis. The structural and energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded conditions, are still debated. Here, we examine the effects of solution crowding on actin polymerization and interpret the results using several crowding...