The transport of proteins between subcellular compartments is a vectorial, energy-requiring process mediated by the budding and fusion of a series of vesicular carriers. As yet, nothing is known of the chemical reactions that underlie these events, or how or in exactly what forms energy is used to sustain such movements. Here we report that fatty acyl-CoA acts as cofactor to a Golgi-associated protein factor (termed NSF) that is required for transport between cisternae of the Golgi stack in a cell-free system. This previously unsuspected connection may offer a link between the complex process of protein transport and a single, well-defined type of chemical reaction. We suggest that an ATP-dependent cycle of fatty acylation and deacylation may play an important role in driving rounds of vectorial protein transport.