A "single-base sequence" is a DNA sequence in which the identities and locations of bases of only one type have been determined. We present experimental procedures for single-base sequencing and describe the effective use of existing software (FASTA) in similarity comparisons of single-base sequences. We determined the theoretical and experimental minimum sequence lengths required for identification of a sequence within a large dataset and optimized the FASTA parameters for use in single-base similarity comparisons. Single-base sequences have been used to identify cDNAs occurring in a database. Single-base sequencing could be used to reduce the redundancy of "shot-gun sequencing."