The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II Δ5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo . Here, we demonstrate that in the context of chromatin, Pol II Δ5 can bind to the c- myc promoter with the same efficiency as wild type Pol II. However, Pol II Δ5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged Δ5 or wildtype Pol II revealed a single, highly mobile Pol II Δ5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex.