Diffusion of solutes and macromolecules in aqueous cellular compartments is required for numerous cellular processes including metabolism, second messenger signaling and protein-protein interactions. The view of the cell interior has evolved from that of a viscous gel to that of a watery but crowded compartment. Recent measurements of fluorescent probe diffusion using photobleaching, correlation microscopy and time-resolved anisotropy methods, have indicated unexpectedly high mobilities of small solutes and macromolecules. This review evaluates experimental evidence defining the rates and barriers for molecular diffusion in cells. Possible implications of regulated molecular diffusion as a rate-limiting step in cell metabolism, and with respect to the delivery of therapeutic agents, are discussed.