Background/Aims: Sexual differences in the transcript levels of various genes including the hepatic isoforms of cytochrome P450 have been extensively studied. Expression of these sexual dimorphic genes have been quantified by Northern blotting, nuclear run on assays and reverse transcriptase-polymerase chain reaction (RT-PCR) methods using numerous housekeeping genes to normalize results. Earlier reports apparently assumed that these internal controls were sex-independent. We have studied sex differences in the expression levels of seven different commonly used housekeeping genes. Methods: We have used quantitative and semiquantitative RT-PCR to monitor the levels of hepatic mRNAs in intact and hypophysectomized male and female rats. Results: We have observed sex-dependent expression of the commonly used housekeeping genes tubulin, cyclophilin, tyrosine aminotransferase, beta-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S and one unconventional housekeeping gene, that is, hypoxia inducing factor-1 alpha, in the livers of intact male and female rats. With the exception of glyceraldehyde-3-phosphate dehydrogenase and 18S which were female-predominant (P < 0.01), the five other genes were found to be expressed at significantly (P < 0.01) higher concentrations in the livers of intact male rats. Similar to findings in which hypophysectomy eliminates sexual dimorphisms in cytochromes P450 expression, of the five housekeeping genes examined, cylophilin, tyrosine aminotransferase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and 18S, all lost their sex-dependent expression following pituitary ablation. Conclusionds: Our data suggest that expression levels of these commonly measured housekeeping genes (structural and metabolic) are not constant, but rather are directly or indirectly regulated by sex-dependent hormones, compromising their application as normalizing controls. (c) 2005 Blackwell Publishing Asia Pty Ltd.