The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. We have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B , is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5′ regions of the five human amylase genes contain a processed γ-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3′ untranslated region of γ-actin. In addition, insertion of an endogenous retrovirus has interrupted the γ-actin pseudogene in four of the five amylase genes.