Hepatic lipase (HL) is present not only in liver, but also in steroidogenic organs, where it is thought to mediate cellular uptake of plasma cholesterol. In rat adrenals and ovaries, the HL gene is transcribed into a variant messenger RNA (mRNA) that lacks exons 1 and 2. Treatment of male Wistar rats with corticotropin resulted in a transient 9-fold increase in the variant HL mRNA in the adrenals, which was paralleled by synthesis of 47- to 49-kilodalton HL-related proteins. In contrast, a delayed, but sustained, 6-fold increase in adrenal HL activity was observed. This difference in time course suggests that the HL activity does not reflect HL-like proteins expressed from the variant mRNA. By Northern blotting, the variant HL mRNA was 2.6 kilobase. By screening a rat genomic library, the 5' end of the variant HL mRNA was located in intron 2 immediately upstream of exon 3. Primer extension analysis mapped the 5' end at nucleotide 465 upstream of exon 3. In promoter-reporter assays, the intron 2 region (-233/+350 with respect to the putative start site) showed no apparent basal activity in HepG2 hepatoma and NCI-H295R adrenocortical cells. The putative promoter in intron 2 was up-regulated in NCI-H295R human adrenocortical cells by treatment with 8-bromo-cyclic adenosine monophosphate. We conclude that intron 2 of the rat HL gene has an alternative promoter with low activity in adrenals, ovaries, and liver. In rat adrenals, this promoter is transiently activated by corticotropin.