The general transcription factor TFIIE plays essential roles in transcription by RNA polymerase II (PolII). Despite recent progress, the elucidation of its precise mechanisms including biological functions awaits further characterization. We report the isolation and characterization of Schizosaccharomyces pombe TFIIE (spTFIIE). Like human and other eukaryotic TFIIE proteins, spTFIIE consists of alpha and beta subunits and the genes encoding both subunits are essential for viability. Chromatin immunoprecipitation assays demonstrated that spTFIIE localizes to promoters in vivo. Mutational analysis of the C-terminal basic helix-loop region of TFIIEbeta, which is involved in the transition from transcription initiation to elongation, revealed that transcription-defective mutants affected in this region are also cold sensitive. The spTFIIEbeta subunit binds both spTFIIEbeta and spTFIIEalpha but spTFIIEalpha binds only spTFIIEbeta. These results indicate that TFIIE forms an alpha2beta2 heterotetramer in which two alphabeta heterodimers are connected via beta subunits. Further analysis of binding specificities showed that spTFIIEbeta binds the Rpb2 and Rpb12 subunits of PolII, whereas spTFIIEalpha predominantly binds Rpb5, which is located at the clamp region and changes conformation upon transcription initiation.