In spite of the important regulatory functions of antisense transcripts in gene expression, it remains unknown how antisense transcription is initiated. Recent studies implicated RNA polymerase II in initiation of antisense transcription. However, how RNA polymerase II is targeted to initiate antisense transcription has not been elucidated. Here, we have analyzed the association of RNA polymerase II with the antisense initiation site at the 3' end of the GAL10 coding sequence in dextrose-containing growth medium that induces antisense transcription. We find that RNA polymerase II is targeted to the antisense initiation site at GAL10 by Reb1p activator as well as general transcription factors (e.g., TFIID, TFIIB, and Mediator) for antisense transcription initiation. Intriguingly, while GAL10 antisense transcription is dependent on TFIID, its sense transcription does not require TFIID. Further, the Gal4p activator that promotes GAL10 sense transcription is dispensable for antisense transcription. Moreover, the proteasome that facilitates GAL10 sense transcription does not control its antisense transcription. Taken together, our results reveal that GAL10 sense and antisense transcriptions are regulated differently and shed much light on the mechanisms of antisense transcription initiation.