N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of approximately 30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-gamma-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation.