In experiments on frog cutaneus pectoris muscle, we established that caffeine (5 mM) treatment in presence of bivalent cations in extracellular solutions (Ca, Sr, Ba or Cd) or in Ca-free solutions led to a rapid long-lasting increase of the frequency of miniature end-plate currents due to rise of intraterminal calcium concentration ([Ca2+]i). Five min exposure of the preparation to caffeine in presence of fluorescent dye FM 1–43 led to the successful staining of nerve terminals. The exposure of preparation to caffeine in Ca-free solutions caused the failure of FM 1–43 loading and increase in nerve terminal width. This inhibition of endocytosis of synaptic vesicles is reversible. We conclude that the presence of Ca2+ in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles. The effect of extracellular Ca2+ on endocytosis might act by non-specific interaction with membrane lipids.