The cytosolic aspartate aminotransferase (cAspAT) is a ubiquitous enzyme that displays liver-specific hormonal regulation. In the hepatoma cell line Fao, both the activity and the mRNA level of cAspAT are increased by glucocorticoids. This effect is potentiated by cAMP and inhibited by insulin. Using in vivo run-on experiments, we showed that these effectors act at the transcriptional level. A cAspAT gene fragment containing 2405 bp of the promoter was sequenced. Deletion fragments of this promoter were inserted upstream of the CAT gene, and the regulation of their activity was assayed following transfection in Fao cells. Stable transfection experiments established that the construct including the entire 2.405-kb fragment undergoes positive regulation by glucocorticoids and cAMP and negative regulation by insulin similar to the regulation of the endogenous gene. A physical separation of the positive and negative control elements is suggested by the fact that cAMP acted on the -682/-26-bp fragment (a 2-fold increase of the stimulation by dexamethasone), whereas the negative regulation by insulin (50% of the stimulation by dexamethasone) required the -1983/-1718-bp fragment. Both regions were required for maximal glucocorticoid activity (6-9-fold increase of CAT activity). We conclude that at least two regulatory regions, a proximal and a distal one, are required for full hormonal regulation of the cAspAT gene.