The acquisition of an efficient stomach lysozyme is associated with the success of the ruminants. Advanced ruminants, such as cow, sheep, and deer, have approximately 10 lysozyme genes, some of which are expressed and function in the stomach and some which are expressed and function in nonstomach tissues (e.g., trachea or kidney). The pig possesses a single conventional lysozyme c gene that is expressed in both stomach and nonstomach tissues, and in this respect is similar to what was thought to exist in the early artiodactyl, before the acquisition of the ruminant lifestyle. To better understand the genetic events that occurred early in the origin and evolution of stomach lysozyme, we have isolated and characterized the pig lysozyme gene. The pig lysozyme gene is similar in size to that of other mammalian species, and both stomach and nonstomach expression utilize the same promoter. All the duplications of the ruminant lysozyme gene occurred after the divergence of the pig lineage from the lineage leading to the advanced ruminants. Comparison of the nucleotide sequence of the coding region of mature stomach lysozymes from advanced ruminants and pig revealed no change in the rate of synonymous substitutions. Comparison of the numbers of nonsynonymous and synonymous substitutions provides evidence for positive selection along the early ruminant lineage. These results indicate that changes in selective pressure, and not mutation rate, account for the changes in rates of stomach lysozyme evolution.