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The ability to perform pairwise sequence alignments and interpret the results from such analyses has become commonplace for nearly all biologists, no longer being a technique employed solely by bioinformaticians. With time, these methods have undergone a continual evolution, keeping pace with the types and scale of data that are being generated both in individual laboratories and by systematic, organismal sequencing projects. As with all computational techniques, the reader should have a firm grasp of the underlying algorithm, always keeping in mind the algorithm's capabilities and limitations. Intelligent use of the tools presented in this chapter can lead to powerful and interesting biological discoveries, but there have also been many cases documented where improper use of the tools has led to incorrect biological conclusions. By understanding the methods, users can optimally use them and end up with a better set of results than if these methods were treated simply as a “black box.” As biology is increasingly undertaken in a sequence-based fashion, using sequence data to underpin the design and interpretation of experiments, it becomes increasingly important that computational results, such as those generated using BLAST and FASTA, are cross-checked in the laboratory, against the literature, and with additional computational analyses to ensure that any conclusions drawn not only make biological sense but also are actually correct.