A central goal of evolutionary biology is to understand the genetic and functional mechanisms through which organisms become adapted to their environments. Testing specific hypotheses about effects of historical genetic changes on organismal functions and fitness, however, has been a major challenge in the field of evolutionary genetics since its inception. This dissertation begins with a brief review covering the history of and historical challenges in evolutionary studies of genetic adaptation. The subsequent chapters report findings from new experimental investigations, using recently developed methods in molecular biology and biochemistry, of two iconic hypotheses of molecular adaptation concerning evolution of the fruitfly alcohol dehydrogenase enzyme (ADH) between and within species. In both of these cases, the results show how historical evolution of ADH happened differently than predicted and highlight the importance of experimentally testing adaptive hypotheses. Further, this work establishes a method—reconstruction of ancestral genes and tests of their biochemical functions in vitro and physiological functions in vivo using transgenic organisms—that allows testing of previously untestable adaptive hypotheses. The last chapter reports emerging themes in protein evolution about the number and biochemical nature of genetic changes involved in the origin and differentiation of molecular specificity.