P-glycoprotein (Pgp) is a small family of plasma membrane proteins, which are capable of transporting substrates across cell membranes. Class I and 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 regulation of all three Pgp genes is still poorly understood. For instance, it is not clear if the three Pgp genes are co-regulated or differentially regulated by external stimuli. This study examined the effect of drugs and collagen sandwich system on expression and transcription of all the three Pgp genes in primary rat hepatocytes. Consistent with previous findings, dramatic overexpression (25-fold) of Class II Pgp mRNA was seen, upon culturing of hepatocytes onto a single layered collagen gel. Hepatocytes sandwiched between two layers of collagen gel exhibited decreased (4.5-fold) Class II Pgp mRNA expression as compared to the single layer system. Treatment of hepatocytes cultured on the single layer collagen system with cytoskeletal disrupting (cytochalasin D, colchicine) but not cytoskeletal stabilizing (phalloidin, taxol) drugs, suppressed Class II Pgp expression. In all cases, no change in Class II Pgp transcription was observed as demonstrated by nuclear run-on studies. This suggests that collagen configuration and drugs affect Class II Pgp mRNA expression predominantly through post-transcriptional mechanisms. In contrast, parallel increases in mRNA expression and transcription of Class I Pgp gene were observed upon culturing of hepatocytes, in the collagen sandwich system, and treatment with some drugs (cytochalasin D, colchicine, and phalloidin). This suggests that Class I Pgp gene is regulated primarily via transcriptional mechanisms by these stimuli. On the other hand, Class III Pgp gene appears to be post-transcriptionally co-regulated with Class II Pgp gene by treatment with the drugs, while collagen configuration affected both transcription and post-transcription of Class III Pgp gene. Finally, dose-dependent studies using cycloheximide provided further evidence that the two MDR-associated genes are not co-regulated. This study has implications for future studies on the molecular mechanisms of Pgp gene regulation.