The roles of three TATA binding protein (TBP) homologs (TBP1, TBP2, and TBP3) in the archaeon Methanosarcina acetivorans were investigated by using genetic and molecular approaches. Although tbp2 and tbp3 deletion mutants were readily obtained, a tbp1 mutant was not obtained, and the growth of a conditional tbp1 expression strain was tetracycline dependent, indicating that TBP1 is essential. Transcripts of tbp1 were 20-fold more abundant than transcripts of tbp2 and 100- to 200-fold more abundant than transcripts of tbp3, suggesting that TBP1 is the primary TBP utilized during growth. Accordingly, tbp1 is strictly conserved in the genomes of Methanosarcina species. Deltatbp3 and Deltatbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the Deltatbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted Deltatbp3 cells exhibited growth rates, final growth yields, and lag times that were significantly reduced compared with those of the wild type when the organisms were cultured with growth-limiting concentrations of acetate, and the acetate-adapted Deltatbp2 strain exhibited a final growth yield that was reduced compared with that of the wild type when the organisms were cultured with growth-limiting acetate concentrations. DNA microarray analyses identified 92 and 77 genes with altered transcription in the Deltatbp2 and Deltatbp3 strains, respectively, which is consistent with a role for TBP2 and TBP3 in optimizing gene expression. Together, the results suggest that TBP2 and TBP3 are required for efficient growth under conditions similar to the conditions in the native environment of M. acetivorans.