Transcription of poly(dA) x poly(dT)-containing sequences was investigated in vitro using plasmids carrying a (dA)34 x (dT)34 tract in the coding region of the lacZ gene. The efficiency of transcription of the (dT)34 sequence on the transcribing strand by Escherichia coli RNA polymerase was substantially lower (approximately 60%) than that of the (dA)34 sequence or of the control lacZ gene. Analysis of the transcription process of the (dT)34 sequence by T3 RNA polymerase showed that the transcription was frequently arrested or terminated at the middle as well as immediately proximal of the (dA)34 x (dT)34 tract, and it occurred more prominently following accumulation of transcription products. This inhibition was strongly enhanced by the addition of the oligonucleotide (dT)34 or poly(U) to the reaction mixture, while (dA)34 and the duplex (dA)34 x (dT)34 suppressed the inhibition. A similar transcriptional inhibition was also observed in transcription mediated by T7 RNA polymerase and eukaryotic RNA polymerase II. We also demonstrated RNA x DNA complex formation of the (dA)34 x (dT)34 tract with poly(U), but not with poly(A). These findings strongly suggest that poly(dT)-containing template sequences interact and form a complex with its transcription products, possibly an RNA x DNA triplex, which blocks further transcription. This would explain the instability of the plasmids transcribing mRNAs with poly(U) but not poly(A) tracts and the underrepresentation of poly(U) but not poly(A) tracts in mRNAs.