PfTBP is a transcriptional factor required by all three types of RNA polymerases in eukaryotic cells. In order to obtain a specific monoclonal antibody (MAb) against PfTBP, a DNA fragment of 684 base pairs (bp) that contained the complete PfTBP gene was amplified by polymerase chain reaction (PCR) and inserted into the pGEX prokaryotic expression vector. The recombinant protein (GST-PfTBP) was expressed in Escherichia coli, purified, and used as antigen to immunize mice. MAbs against PfTBP were obtained and hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Western blotting and immunofluorescence assays showed that MAb Pf.r1 recognized the PfTBP protein in nuclear extracts from Plasmodium falciparum as well as a native protein in the nuclei of this parasite. This MAb will be a helpful tool for the identification of the TBP associated factors (TAFs), which are apparently highly divergent with other eukaryotes. This information could help to identify new candidate gene products to develop novel drugs or vaccines.