We have isolated and characterized single-amino-acid substitution mutants of RNA polymerase alpha subunit defective in CAP-dependent transcription at the lac promoter but not defective in CAP-independent transcription. Our results establish that (1) amino acids 258-265 of alpha constitute an "activation target" essential for CAP-dependent transcription at the lac promoter but not essential for CAP-independent transcription, (2) amino acid 261 is the most critical amino acid of the activation target, (3) amino acid 261 is distinct from the determinants for alpha-DNA interaction, and (4) the activation target may fold as a surface amphipathic alpha-helix. We propose a model for transcriptional activation at the lac promoter that integrates these and other recent results regarding transcriptional activation and RNA polymerase structure and function.