Mutagenesis is a change or alteration in the DNA sequences of a gene. Mutagenic events may occur spontaneously within the genome of an organism and many times do not lead to functional consequences or an altered phenotype. However, at times a change in the coding sequence of a gene manifests itself as a dysfunctional phenotypic trait or predisposes an individual to a particular disease. The human genome may contain variations or mutations in an individual or in a related group of individuals that predispose them to a particular disease. Identifying and understanding these mutations (or polymorphisms) in gene structure can aid in the understanding, diagnosis, and treatment of patients. Quite the opposite to the mutations that spontaneously occur and may be difficult to identify, purposefully introducing mutations into a candidate gene that is then expressed in a cell or mouse model systems can readily reveal important information. This chapter provides an overview of the strategy to clone a gene, express it in a cultured cell system, and elucidate the function of the expressed gene product using mutagenesis methods. The value of this type of experimental approach will be highlighted by reviewing two pulmonary genes that have been analyzed using in vitro mutagenesis methods, the potassium channel Kv1.5 and the bone morphogenetic protein receptor II.