The -10 and -35 regions of E. coli promoter sequences are separated by a spacer region which has a consensus length of 17 base-pairs. This region is thought to contribute to promoter function by correctly positioning the two conserved regions. We have performed a statistical evaluation of 224 spacer sequences and found that spacers which deviate from the 17 base-pair consensus length have nonrandom sequences in their upstream ends. Spacer regions which are shorter than 17 base-pairs in length have a significantly higher than expected frequency of purine-purine and pyrimidine-pyrimidine homo-dinucleotides at the six upstream positions. Spacer regions which are longer than 17 base-pairs in length have a significantly higher than expected frequency of purine-pyrimidine and pyrimidine-purine hetero-dinucleotides at these positions. This suggests that the nature of the purine-pyrimidine sequence at the upstream end of spacer regions affect promoter function in a manner which is related to the spacer length. We examine the spacer sequences as a function of spacer length and discuss some possible explanations for the observed relationship between sequence and length.