The injectisome is the structural core of the membrane-embedded type III secretion system (T3SS), and bacterial effector proteins are secreted through this complex into the host. Radics et al. used cryo-electron tomography to visualize individual Salmonella enterica serovar Typhimurium T3SSs loaded with GFP-fused substrates that were transported into the injectisome but that subsequently became trapped and blocked the secretion of other substrates. Structural studies of substrate-trapped complexes showed that transport occurs in a polarized manner, with the amino-terminal domain of the substrate entering the complex first. Characterization of the secretion path within the injectisome suggested that T3SS effectors are transported in an unfolded state.