In this report we describe the cloning and initial characterization of TAF40, a gene that encodes a yeast TATA-binding protein-associated factor (yTAF) of Mr = approximately 40,000. This gene has many similarities to other yTAFs described thus far in that it is present at a single copy per haploid genome, it is essential for viability, and the deduced protein sequence of yTAF40 exhibits similarity to previously described human and Drosophila TAFIIs. Immunological studies confirm that yTAF40 protein is a subunit of a large multiprotein TATA-binding protein-TAF complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Transcription reactions performed using yeast whole cell extracts reveal that of the three nuclear RNA polymerases only RNA polymerase II function is abrogated when yTAF40 and associated proteins are immunodepleted from solution, indicating that the functionality of the multiprotein complex containing yTAF40 is RNA polymerase II-specific. By these criteria yTAF40 appears to encode a bona fide RNA polymerase II-specific TAF, and thus the protein that it encodes has been termed yTAFII40.