Amylase transcription in the human salivary gland results from the evolutionary juxtaposition of two inserted elements, a gamma-actin pseudogene and an endogenous retrovirus, to create an unusual salivary-specific promoter. We utilized these structures as molecular tags to characterize the amylase genes in extant primates by polymerase chain reaction amplification of promoter fragments from genomic DNA. Six distinct amylase promoter structures were identified, which allowed us to infer the structures of common ancestors and trace the evolution of the modern human amylase promoters. Our data show that integration of the pseudogene and retrovirus were evolutionarily recent events. The gamma-actin pseudogene integrated after the divergence of the New World monkeys from the primate ancestral tree, and the retrovirus integrated later, after the divergence of the Old World monkeys. The New World monkey amylase promoter represents the mammalian amylase precursor structure before integration of the two retroposons. Two distinct amylase genes were identified in the Old World monkeys, one with a complete gamma-actin pseudogene insert and another novel structure with a truncation of the gamma-actin sequences. We demonstrated abundant amylase expression in the saliva of an Old World monkey, indicating that the endogenous retrovirus is not required for amylase transcription in the primate salivary gland.