The neuromuscular-blocking effects of neomycin, streptomycin, and gentamicin were examined in fast-and slow-twitch muscles of pentobarbital-anesthetized cats. These antibiotics were more effective in reducing indirectly stimulated contractions of the gastrocnemius muscle than of the ipsilateral soleus muscle in adult cats. Respiratory-depressant effects of these antibiotics were more closely associated temporally with effects on soleus than on gastrocnemius twitch. Neomycin was a more potent neuromuscular blocking agent in kittens > 7 weeks old than in kittens < 6 weeks old. Calcium ions were more effective in antagonizing the effects of neomycin on gastrocnemius than on soleus twitch; however, neostigmine was more effective in antagonizing the effects of neomycin on soleus than on gastrocnemius twitch. These data indicate a difference in responsiveness of fast- and slow-twitch muscles (fast > slow) to the neuromuscular-blocking effects of aminoglycoside antibiotics, and suggest that this may be related to a difference in the predominance of pre- and postjunctional inhibitory actions of these agents in the 2 muscle types.