Visceral toxicosis of catfish (VTC) is a syndrome characterized by sudden mortality in apparently healthy market- and brooder-sized catfish (Ictalurus punctatus). This paper reports the design of a catfish neutralization assay to detect botulinum in catfish with VTC and verification by Endopep mass spectrometry (Endopep-MS). Sera from 6 affected catfish were incubated with botulinum antitoxin serotypes A, B, C, D, E, or F. For each serum sample, 3 experimental fingerlings were injected intracoelomically with each serotype-serum mixture and placed separately in an aquarium. Three fish were injected with VTC-affected serum only, and 3 fish were injected with unaffected serum only and also placed in separate aquaria. Signs of morbidity and mortality were seen in fish injected with sera combined with serotype A, B, C, or D, as well as in positive controls. No morbidity or mortality was seen in fish injected with sera combined with antitoxin serotypes E or F or negative control serum. Sera from affected and unaffected catfish were sent to Centers for Disease Control and Prevention for detection and differentiation of botulinum neurotoxin. Aliquots of 0.5 ml of sera were incubated with magnetic beads coated with antibodies to botulinum, and the beads were subjected to the Endopep-MS reaction. Sera from affected catfish tested positive for botulinum E. Sera from 34 unaffected catfish tested negative for botulinum. Although there was not enough botulinum present in affected samples to obtain exact quantification, the estimated quantity of botulinum E in these sera samples was between 0.01 and 0.5 mouse LD50/ml.