Primers, based on the cDNA nucleotide sequences for rat hepatic squalene synthase (EC 2.5.1.21) (McKenzie, T.L., Jiang, G., Straubhaar, J.R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374), were synthesized and used for the amplification and sequencing of a 1672-base pair (bp) cDNA for the human hepatic squalene synthase (HSS) from human hepatic RNA. An open reading frame of 1251 bp encoding 417 amino acids (M(r) = 48,200) was detected for HSS. We have constructed a pHSS 1286 expression vector by molecular cloning of a 1286-bp cDNA, that includes sequences of the entire coding region for HSS, into pBluescript. Expression in Escherichia coli of a functional, full-length HSS was confirmed by immunoblot analysis and enzymatic activity. Northern blot analyses of poly(A+) RNA obtained from the human hepatoma cell line HepG2 show three distinct size classes of mRNA for HSS. 1.4-, 1.6- and 2.1-kilobase mRNA were observed. The relative abundance is in the order 1.6 > 1.4 > 2.1 and did not change when the cells were grown in the presence of 25-hydroxycholesterol or lovastatin. The ratio between the level of HSS mRNA in cells grown in the absence and presence of 5 micrograms/ml 25-hydroxycholesterol varies between 8- and 16-fold. This lowering of the mRNA level was observed when the cells were grown in 10% of either full serum or lipid-depleted serum. A 2.7- and 4.0-fold increase of HSS mRNA was observed when HepG2 cells were grown in the presence of 5 micrograms/ml lovastatin in lipid-depleted or full serum, respectively. These studies show that HSS exhibit a relatively high level of transcriptional regulation in response to 25-hydroxycholesterol regardless of the presence of cholesterol in the growth media.