Western blotting of the insulin-secreting beta-cell lines HIT-15 and RINm5F with anti-SNAP-25 (synaptosomal associated protein of 25 kDa), anti-synaptobrevin, and anti-syntaxin 1 antibodies revealed the presence of proteins with the same electrophoretic mobility as found in neural tissue. Permeabilization of both of these insulinoma cell lines to botulinum neurotoxin A by electroporation resulted, after 3 days of culture, in the loss of approximately 90% of SNAP-25 immunoreactivity. A similar permeabilization of these cells with botulinum neurotoxin B resulted in the cleavage of approximately 90% of the synaptobrevin-like immunoreactivities. Botulinum neurotoxin F also cleaved approximately 90% of the synaptobrevin-like immunoreactivity in RINm5F cells. The permeabilization of both insulinoma cells to neurotoxin A resulted in a > 90% inhibition of potassium-stimulated, calcium-dependent insulin release. By contrast, permeabilization of the insulinoma cell lines to neurotoxin B resulted in only a approximately 60% inhibition of potassium-stimulated insulin release in HIT-15 cells, and neither neurotoxin B nor F caused inhibition in RINm5F cells. Thus HIT-15 and RINm5F cells contain the components of the putative exocytotic docking complex described in cells derived from the neural crest. In HIT-15 cells both SNAP-25 and synaptobrevin appear to be involved in calcium-dependent insulin secretion, whereas in RINm5F cells SNAP-25 but not synaptobrevin is involved.