A C.G to A.T transversion at position +10 of the lac promoter activates a nascent sigma 70-dependent promoter (the +10A promoter). The lac +10A promoter has two unusual properties; it programs a large family of transcripts with multiple 5' ends, and its sequence bears little resemblance to other sigma 70-dependent promoters. The 5' end of the +10A in vivo mRNA was determined to contain oligo(U) sequences of varying lengths suggesting that the true start site was at a run of three T.A base-pairs located 20 to 22 bp downstream of the lac wild-type promoter start site, and that the transcription initiation process involved a transcriptional slippage event (which resulted in multiple rU incorporation). Only mutations at or near the start site and those deletions that changed the location of the start site abolished this transcriptional slippage property of the transcription initiation process. This transcriptional slippage was also found to be promoter independent because changing the lac UV5 start site to a run of five T.A base-pairs (-1 to +4) resulted in similar transcriptional slippage. Saturated mutagenesis of the +10A promoter identified a potential -10-like region and indicated that sequences immediately upstream of the -10 region contributed to the promoter's activity. Decreasing the weak -35 region homology did not change promoter strength; however, introduction of the consensus -35 hexamer TTGACA increased expression tenfold. RNA polymerase bound to the +10A promoter partially protects a 20 base-pair sequence from DNase I digestion upstream of the start site. These results suggest that RNA polymerase interacts with the +10A promoter in a different manner from that for the majority of sigma 70 promoters.