Previous work from this laboratory has shown that glucocorticoids and (Sp)-cAMPS, a cyclic AMP analogue, stimulate the accumulation of angiotensinogen mRNA in isolated hepatocytes. The present study demonstrated that glucocorticoids stimulate the accumulation of a novel, 2.25-kilobase transcript of the angiotensinogen gene, both in isolated hepatocytes and in the intact liver. (Sp)-cAMPS was synergistic with dexamethasone in causing the accumulation of the larger species of RNA in hepatocytes, but had no effect by itself. Primer extension analysis and S1 mapping experiments proved that the 2.25-kilobase angiotensinogen RNA consisted of two larger forms of angiotensinogen RNA extended at their 5' ends. The novel transcripts are generated by the use of two new transcription initiation sites in the angiotensinogen gene, located at nucleotide positions -328 and -386 relative to the start site at position +1 used in the absence of hormone. A consensus TATA box is found at the expected position 25-30 nucleotides upstream from the start site at position -328, and another TATA-like sequence is found 25-30 nucleotides upstream from the start site at -386. In addition, two consensus glucocorticoid response elements occur upstream from the initiation sites. The larger angiotensinogen RNAs do not appear to code for a novel form of the angiotensinogen protein. It appears that glucocorticoids can direct transcription from a second promoter in the angiotensinogen gene and that this promoter is absolutely dependent on the hormone. To date, this situation appears to be unique to the angiotensinogen gene.