Nerve-stimulated fade in muscle is generally accepted as a prejunctional phenomenon mediated by block of prejunctional acetylcholine receptors (AChRs) at the nerve terminal, whereas decrease of twitch tension is considered a postjunctional effect due to block of muscle AChRs. Using ligands with specific pre- or postjunctional effects only, we tested the hypothesis that fade is not necessarily a prejunctional phenomenon. Neuromuscular function in rats was evaluated after IM (2.5 U) or IV (12.0 U) injection of botulinum toxin (Botx), or IV (250 μg/kg) α-bungarotoxin (α-BTX) alone. The acute neuromuscular effects of IV 2 mg/kg dihydro-β-erythroidine (DHβE), alone and in combination with α-BTX, were also tested. Botx decreases vesicular release of ACh, and α-BTX binds to postjunctional nicotinic AChRs only, whereas DHβE binds specifically to prejunctional α3β2 AChRs only. In view of the lack of acute effects of Botx even at 2 hours after IV injection, its neuromuscular effects were also evaluated at 24 hours after IM injection (0.6 U) and compared with IM injection of α-BTX (25 μg/kg) or saline also given 24 hours earlier. The sciatic nerve-tibialis muscle preparation, during train-of-four and tetanic stimulation, was used to test neuromuscular effects in vivo. IV and IM Botx had no observable neuromuscular effects at 2 hours. IV α-BTX caused twitch depression within a few minutes, and significant fade (P = 0.002) at 75% of baseline twitch tension; these effects persisted until the end of the observation period of 2 hours. IV DHβE alone caused no significant change in single twitch (P = 0.899) or train-of-four ratio (P = 0.394), but significantly enhanced the fade of IV α-BTX (P = 0.001 at 75% of baseline twitch tension). IM Botx or α-BTX, at 24 hours after their injection, resulted in a significant decrease of single twitch and tetanic tensions (P < 0.0001), but Botx did not cause fade, whereas α-BTX caused significant (P < 0.0001) fade at 24 hours. The tibialis muscle weights and protein expression of α1 subunit of AChR (Western blots) did not differ between Botx, α-BTX and saline-injected groups at 24 hours but increased in denervated muscle (positive control). Botx-induced decreased ACh release in and of itself does not cause fade but does cause decrease of absolute tensions. Decrease of available (functional) postjunctional AChRs by α-BTX did induce fade. The prejunctional fade effects of DHβE on α3β2 AChRs become manifest only when the margin of safety was decreased by concomitant administration of α-BTX. Thus, fade during repetitive stimulation is not always a prejunctional phenomenon and may also reflect the decreased margin of safety of neurotransmission, which can be due to a pure postjunctional AChRs block or to a combination of both pre- and postjunctional AChRs block. Block of prejunctional α3β2 AChRs alone is not necessary and sufficient to cause fade.