In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3'-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3'-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3'-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3'-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3'-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis.