A monoclonal antitoxin/colloidal gold probe and sequential centrifugation were used to study synthesis, translocation and export of Clostridium botulinum strain 62A neurotoxin (NT). Exponential growth occurred after 5 h of anaerobic incubation of spores and continued for 15-16 h. NT was detected at 15 h using the probe and transmission electron microscopy (TEM), 2 h earlier than the first detection by the mouse bioassay. During exponential growth, the probe localized NT primarily in the cytoplasm, on the inner side of the cytoplasmic membrane and in the cell wall. During stationary and death phases, the NT was located within the cytoplasm, cell wall and extracellularly. NT was released from the cell during cell wall exfoliation. Cells retained NT after repeated gelatin-phosphate washes and sequential centrifugations, consistent with the TEM observation that the NT is bound to the cell wall. These observations indicate that the process of Cl. botulinum type A NT production follows a sequence of synthesis, translocation across the cytoplasmic membrane and export through the cell wall.