1. The anionic dye Erythrosin B increases quantal transmitter release from frog neuromuscular synapses. Experiments were performed to determine the role of ions and light in this presynaptic effect. 2. In calcium-free saline containing 1 mM-EGTA, Erythrosin B increased miniature end-plate potential (m.e.p.p.) frequency at a more rapid rate than in normal saline. 3. The dye's effect was influenced by extracellular calcium ions in a complex manner. Dye-induced release was minimal in Ringer solution containing 0.1 mM-calcium, and higher in calcium concentrations above or below 0.1 mM. 4. Erythrosin B-induced spontaneous release also occurred in saline which contained 1 mM-EDTA and was free of both calcium and magnesium ions. 5. Temporary removal of external sodium ions did not alter the progressive increase in m.e.p.p. frequency produced by the dye. 6. Elevation of the potassium concentration of the external medium (from 2 to 20 mM), which presumably depolarized nerve terminals and increased their calcium permeability, did not change the rate of increase of dye-induced release when preparations were in a reversed (outward) electrochemical gradient for calcium ions. 7. A reduction in light intensity of at least six orders of magnitude reduced the effect of Erythrosin B by 50%, suggesting that photoactivation is not the primary basis for the dye's action. 8. These results indicate that Erythrosin B is not acting solely by altering the ionic permeability of the presynaptic nerve terminal to calcium, magnesium, or sodium ions, or by altering the calcium metabolism of the terminal. The enhanced effect of the dye in calcium-free saline suggests that it may be competing with calcium at a common site, while the enhancement of its effect in elevated external calcium suggests that the dye may also increase the permeability of the nerve terminal to calcium ions.