Peripherin is a type III neuronal intermediate filament protein detected within the intraneuronal inclusions characteristic of amyotrophic lateral sclerosis. The constitutively expressed peripherin isoform is encoded by all nine exons of the human and mouse peripherin genes to generate a protein species of approximately 58 kDa on sodium dodecyl sulfate-polyacrylamide gels. Expression of this isoform, termed Per-58, generates a filament network in transfected SW13 vim cells. On immunoblots of cell lysates derived from these transfected cells, we have consistently observed a second peripherin species of approximately 45 kDa. In this study, we show that this species is a novel peripherin isoform generated through the use of an in-frame downstream initiation codon. This isoform, that we have designated Per-45, is co-expressed together with Per-58 and, thus, constitutive in both human and mouse. Using mutational analysis, we show that Per-45 is required for normal network formation, with the absence of Per-45 leading to irregular filamentous structures. We further show that peripherin expression in the normal nervous system is characterized by tissue-specific Per-58 : Per-45 isoform ratios. Taken together, these results identify novel processing requirements for peripherin expression and indicate a hitherto unrecognized role for neuronal intermediate filament network formation through intra-isoform associations.