Guanidine has been shown to potentiate neurotransmission at the skeletal neuromuscular junction. The present study was undertaken to evaluate whether reductions in extracellular calcium affect the action of guanidine on the reversal of neuromuscular blockade induced by dibekacin and d-tubocurarine in the rat phrenic-hemidiaphragm preparation. Guanidine reversed the blocking effect of d-tubocurarine, the maximal effect occurring with 3 mM at 15 min. This reversal effect of guanidine was potentiated when the extracellular calcium concentration was reduced from 2.5 mM to 1 mM and 0.3 mM. Guanidine was also effective in reversing the neuromuscular depression caused by dibekacin. In this case, it was more potent than in the d-tubocurarine-treated preparations. The maximal effect was observed with 3 mM after 15 min of exposure. When the extracellular calcium concentration was reduced from 2.5 mM to 1 mM, a potentiation of guanidine effects was observed. In this situation, the maximal effect of guanidine was observed with only 0.7 mM. However, further reductions of extracellular calcium (0.3 mM) did not show further increases. The present results show that guanidine reverses the blockade induced by dibekacin and d-tubocurarine, being more potent against the former drug. This suggests that guanidine may be useful in relieving the blockade due to a restriction of calcium entry. Its effects were potentiated when the extracellular calcium concentration was lowered to 1 mM and, in d-tubocurarine-treated muscles, to 0.3 mM. Thus, the effects of guanidine are calcium-dependent but only in a certain concentration range.