In order to better understand the role of the -35 sequence motif in transcription initiation by Escherichia coli sigma 70 RNA polymerase holoenzyme, -35 promoter elements of limited nucleotide composition have been selected from random DNA sequences. Functional promoter elements have been identified that are composed of just two nucleotide species (A,T; G,C; T,C; and T,G) and one nucleotide species (poly(dT)). From this study of 81 promoter mutations, two conclusions can be made. First, given a population of random DNA sequences, there exist sequences that form stronger -35 promoter elements than the consensus sequence. These sequences lack some features of the consensus sequence and reveal unexpected homology in the ordinarily nonconserved spacer sequence. Second, the sequence requirements at the -35 site may suggest a possible role for DNA secondary structure in the recognition of promoters by RNA polymerase.