We cloned the rat alpha-amylase gene Amy-1 and compared its structure and expression with its mouse counterpart. The results showed that the general organization of the transcriptionally active rat Amy-1 gene was similar to that of its mouse counterpart; i.e., the rat gene also contained two independent transcriptional promoters. The distance between the two promoters in the rat gene was, however, more than double (6 kilobases) that measured in the mouse gene (2.8 kilobases). In addition, the rat genome also contained an independent, orphonlike version of the weaker Amy-1 promoter, which was transcriptionally silent. In spite of the similar overall organization of the Amy-1 genes in mouse and rat cells, an interesting difference was observed in the expression of the weak promoter in these two closely related rodents. In rats this promoter was significantly active only in liver cells, while in mice it was utilized with similar efficiencies in parotid, liver, and pancrease cells. Moreover, the transcripts produced in rat liver had a very heterogeneous population of 5' ends, located between 180 and 220 nucleotides upstream of the two homologous start sites observed for this promoter in mouse liver, even though the sequences around this region were strongly conserved between the two species.