The components of the cellular machinery that accomplish the various complex and dynamic membrane fusion events that occur at the division plane during plant cytokinesis, including assembly of the cell plate, are not fully understood. The most well-characterized component, KNOLLE, a cell plate-specific soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptor (SNARE), is a membrane fusion machine component required for plant cytokinesis. Here, we show the plant ortholog of Cdc48p/p97, AtCDC48, colocalizes at the division plane in dividing Arabidopsis cells with KNOLLE and another SNARE, the plant ortholog of syntaxin 5, SYP31. In contrast to KNOLLE, SYP31 resides in defined punctate membrane structures during interphase and is targeted during cytokinesis to the division plane. In vitro-binding studies demonstrate that AtCDC48 specifically interacts in an ATP-dependent manner with SYP31 but not with KNOLLE. In contrast, we show that KNOLLE assembles in vitro into a large approximately 20S complex in an Sec18p/NSF-dependent manner. These results suggest that there are at least two distinct membrane fusion pathways involving Cdc48p/p97 and Sec18p/NSF that operate at the division plane to mediate plant cytokinesis. Models for the role of AtCDC48 and SYP31 at the division plane will be discussed.