We have detected formate synthesis by Clostridium thermocellum 27405 cultured in both cellobiose and alpha-cellulose. While formate synthesis has been reported for one strain of C. thermocellum (strain I-1-B), numerous studies of C. thermocellum 27405 fermentation, conducted under different growth conditions, failed to detect the presence of formate. Thus, the status of formate synthesis as a fermentation end product by C. thermocellum has been uncertain. Formate synthesis competes with the synthesis of hydrogen (H2) as a fermentation end product, and thus would negatively impact H2 yields in processes designed to generate H2 from biomass. Understanding the mechanism of formate synthesis is the first step in devising means of mitigating its production. Transcription of putative pfl, fnr, and adhE genes, encoding pyruvate formate-lyase (PFL), PFL-activating enzyme (PFL-AE), and alcohol dehydrogenase E (ADH-E) enzymes, respectively, were detected by reverse transcriptase polymerase chain reactions using total RNA extracted from stationary phase C. thermocellum cultured on cellobiose. The PCR products observed correspond to the expected amplicon sizes. Nucleotide sequence analysis of the cloned PCR products followed by BLAST analyses confirmed their identity. Formate production was detected throughout growth, and PFL enzyme activity was detected in late log and stationary phase (OD600 = 0.7 and 0.9, respectively) in extracts of C. thermocellum cultured on cellobiose. BLAST analyses revealed that C. thermocellum PFL and PFL-AE have greater amino acid sequence identity with equivalent enzymes from Bacillus and Thermocynechococcus species than with other Clostridium species, but C. thermocellum ADH-E has greater amino acid sequence identity with Clostridium species.