Specific initiation of transcription of cloned conalbumin and ovalbumin genes has been obtained in a cell-free system in the presence of purified calf thymus RNA polymerase B. Comparison with the adenovirus-2 early (E1A) and major late genes reveals marked variations in transcriptional efficiencies. Specific fragmentation of the conalbumin gene DNA indicates that a short DNA segment 5' to the initiation site is involved in specific transcription.