We have identified the 5' termini of ovomucoid RNA transcripts isolated from estrogen stimulated chick oviduct by S1-nuclease mapping. Most of the ovomucoid RNA molecules (88-89%) are initiated at a predominant region which is positioned at about 30 basepairs downstream from a "TATATAT" sequence. However, there is at least one additional initiation region which accounts for 9% of the ovomucoid RNA transcripts. It is located at approximately 80 basepairs further upstream (5') from the major initiation region. DNA sequence data reveal that an AT-rich sequence resembling the "TATA" box is also present 30 nucleotides before this minor region. Our finding provides additional insight into the molecular mechanism involved in the transcription initiation of eucaryotic genes, and indicates that in vivo synthesis of mRNA may not require an absolute site for initiation of transcription.