Yeast Ino2p-Ino4p heterodimeric complex is well known as a transcriptional activator for the genes regulated by inositol and choline, such as the INO1 gene. Apl2p is a large subunit of the yeast adaptin complex, an adaptor complex required for the clathrin coat to bind to the membrane. We found that Ino2p, Ino4p, and Apl2p form a ternary complex. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation. Ino2p and Ino4p bind to Apl2p in the same region of Apl2p, viz., at the middle part and the C-terminal part. Ino2p and Ino4p bind to Apl2p independently, but more strongly when both are present. Furthermore, a disruption of APL2 together with INO2 or INO4 rendered yeast cells sensitive to oxidative stress. INO2-APL2 double disruptants also showed growth inability in nonfermentable carbon sources, such as glycerol. These results indicate a genetic interaction between APL2, INO2 and INO4 and uncovere novel functions of the Ino2p-Ino4p-Apl2p complex in yeast.