Transcription of bacteriophage SP01 middle promoters is specifically initiated by a complex of the Bacillus subtilis host's RNA polymerase core (E) with the SP01 gene 28 transcription-regulating protein, gp28. Normal SP01 DNA contains hydroxymethyluracil (hmUra) in place of thymine and E . gp28 preferentially transcribes hmUra-containing DNA. Hybrid DNA molecules containing an SP01 middle promoter, PM25 . 1, have been constructed in which one DNA strand contains T and the other hmUra. The major feature of these reciprocal hybrid promoters is that one has, predominantly, T substituted for hmUra in the central -35 recognition sequence in the transcribed strand, while the other has, predominantly, T substituted for hmUra in the -10 recognition sequence in the non-transcribed strand. Binding by the E . gp28 RNA polymerase and transcription of these hybrid promoters and of the normal, all-hmUra, promoter have been compared. Both hybrid promoters are weaker than the normal PM25 . 1 promoter, but the hybrid promoter with T substituted in the -10 sequence is the weakest of the set. The DNase I footprint of the normal PM25 . 1 promoter shows temperature-dependent protection of a relatively long stretch of DNA downstream from the transcriptional start site, correlating with a thermal transition of transcriptional activity of promoter complexes. The stronger of the hybrid promoters also undergoes this transition, but the weaker does not. We discuss these findings in terms of protein-DNA interactions determining specificity for a modified nucleotide at this promoter.