We have investigated the proteolytic processing of chromogranin A, chromogranin B and NESP55 (a novel chromogranin-like protein) during axonal transport using pig splenic nerve as a model. We have also studied the presence of chromogranin-derived peptides in the perfusate during electrical stimulation of this nerve. High-performance gel filtration chromatography followed by radioimmunoassay (RIA) revealed that chromogranins are proteolytically processed to varying degrees during axonal transport. For chromogranin A and NESP55, the precursor is still present in the proximal part of the nerve, whereas in the distal part and nerve terminals, intermediate-sized peptides and the free peptides GE-25 and GAIPIRRH dominate, respectively. For chromogranin B, the precursor has already been processed to an intermediate-sized peptide in the proximal part of the nerve, which is also present in the distal parts together with the free peptide PE-11. For chromogranin B and NESP55, only the free peptides PE-11 and GAIPIRRH, or in the case of chromogranin A, the free peptide GE-25 plus an intermediate-sized one, are released from the terminals into the splenic perfusate. These results demonstrate that chromogranins are processed to smaller peptides during axonal transport.