Distinct differences exist in the function and regulation of the individual p-glycoprotein (pgp) members in many species. In order to study regulation of individual pgp mRNA isoforms it is, therefore, necessary to have probes that can distinguish between the various pgp isoforms. However, to date few studies examining hepatic gene expression in rat liver have used pgp gene specific probes. Towards this end we screened a cDNA library constructed from a normal rat liver with a human pgp1 cDNA and isolated a partial cDNA for class III pgp, rat pgp3. By comparison of the sequence of this new rat pgp3 cDNA with genomic and cDNA sequences for rat pgp1 and rat pgp2 we selected oligonucleotide probe sequences that would allow us to differentiate between the highly homologous rat pgp2 and pgp3 genes on Northern blots and by polymerase chain reaction (PCR). We found that pgp3, for both male and female rats, was the predominant form of pgp expressed in normal rat liver with males consistently expressing several-fold lower levels of pgp3 than females. Because many genes are zonally expressed in the hepatic acinus we examined the possibility that pgp3 might show heterogeneous distribution as well. We found, by in situ hybridization of paraformaldehyde-fixed rat liver sections that pgp3 was distributed non-uniformly across the hepatic acinus with a gradient that showed the highest expression toward the terminal hepatic venule. We confirmed this finding by selectively isolating hepatocytes from either the terminal hepatic venular or periportal zones using a digitonin/collagenase perfusion procedure. Application of specific pgp3 PCR primers to RNA isolated from hepatocytes from these areas confirmed that pgp3 mRNA was the predominant form in the hepatocytes surrounding the terminal hepatic venule. Finally, we examined pgp3 expression in a variety of tissues by Northern blot analysis and found that pgp3 was most highly expressed in the liver and gastrointestinal tract (with a gradient of expression from small to large intestine), while low levels were found in the kidney, heart and brain. Pgp3 mRNA was undetectable in the adrenal gland and in skeletal muscle. In summary, using rat pgp gene specific oligonucleotide probes we found that pgp3 gene expression is regulated by anatomic location with the highest mRNA expression in organs that are involved in drug detoxification. Our results also demonstrate heterogeneity of hepatic rat pgp3 gene expression, which is influenced by both gender and by acinar location.