During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes,KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion.KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 andsec72Δ, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Δ andsec72Δ, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 andkar5 mutations were suggestive of protein–protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but notSEC63) strongly suppressed the mating defect ofkar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8mutant zygotes revealed a nuclear fusion defect different fromkar2, kar5, and kar7/sec71mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.