P-glycoprotein (Pgp) are a small family of plasma membrane proteins capable of transporting substrates across cell membranes. Class I and class II Pgp are able to transport drugs and have been shown to mediate multidrug resistance (MDR). Class III Pgp is a long chain phospholipid transporter and does not mediate MDR. The expression and regulation of Pgp genes in animal tissues are not well understood. In this study, the protein synthesis inhibitor cycloheximide was used as a tool to understand Pgp gene expression and regulation in animal tissues. The sensitive RNase protection assay was used to detect changes in Pgp mRNA levels and nuclear run-on assay was used to determine whether transcription or post-transcription is important. The results showed that cycloheximide significantly induced class II Pgp expression in all tissues examined. This was predominantly through post-transcriptional effect. In contrast, the relatively modest increase in class I Pgp expression by cycloheximide was found to be mainly due to increased transcriptional activity. On the other hand, cycloheximide induced class III Pgp expression in some tissues while caused decay of class III Pgp mRNA in other tissues. The transcriptional and post-transcriptional mechanisms exerted by cycloheximide on Pgp genes are discussed. These findings have implications for our understanding of gene regulation in animal tissues and MDR reversal strategies in vivo.