Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbo(ts)) displays paralysis within minutes following a temperature shift from 25 degrees C to 37 degrees C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbo(ts) displays a strong synergistic interaction with the Syntaxin-1A TS allele syx(3-69), recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbo(ts) showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbo(ts);syx(3-69) double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbo(ts) synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbo(ts) displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbo(ts) synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.