Ubc9p is the sole E2˗conjugating enzyme for SUMOylation and its proper function is required for regulating key nuclear events such as transcription, DNA repair and mitosis. In Tetrahymena thermophila the genome is separated into a diploid germline micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both the MIC and MAC genome is lethal. Rescue of the lethal phenotype with a GFP˗UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl2˗dependent expression of GFP˗Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs but MACs continued to divide. Unequal partitioning of MIC DNA was observed in cells undergoing mitosis. Interestingly, expression of a catalytically inactive Ubc9p resulted in the accumulation of multiple MICs but no defect in cytokinesis was observed. During sexual reproduction endogenously tagged Ubc9p localized in the parental MAC at early stages of conjugation and later accumulated in the developing MAC. The timing and localization is consistent with our previous observations that a large increase in SUMOylation occurs in the developing MAC between 7-10 hours post mixing. Similar to Uba2p (SUMO-activating enzyme) depleted cell lines, Ubc9p depleted cell lines cannot form cell pairs after nutrient starvation and fail to mate. To analyze the function of Ubc9p during sexual reproduction we expressed the catalytically inactive Ubc9p in mating cells. A large fraction of mating pairs (42%) arrested at an early stage of macronuclear development prior to the elimination of the parental MAC. This arrest was dependent on induced expression of the mutant Ubc9p and is consistent with our observation that Ubc9p is localized in the developing macronucleus. The results demonstrate the importance of Ubc9p and the SUMO pathway in the sexual life cycle of Tetrahymena.