The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs. Here, we report the isolation and preliminary characterization of cryopreserved hepatocytes from male PXR and CAR knockout (SD) rats and PXR/CAR double knockout (SD) rats (Horizon Discovery - SAGE Labs). We were successful in the isolation and cryopreservation of the hepatocytes from the three knockout models, yielding hepatocytes with high (>80%) viability and plateability. The cryopreserved hepatocytes from wildtype and knockout rats were cultured for the evaluation of gene expression in the presence and absence of PXR and CAR ligands. The cryopreserved hepatocytes were recovered using UCRM (IVAL Inc.) and plated in 24-well collagen-coated plates. The hepatocytes formed near confluent monolayer cultures with epithelial morphology typical of primary cultured rat hepatocytes. After culturing overnight, medium was changed to protein free induction medium for rat hepatocytes (RHIM, IVAL Inc.). The hepatocytes were treated the next day with the PXR-ligand pregnenlone-16α-carbonitrile (PCN) and the CAR-ligand 3,3′,5,5′-tetrachloro-1,4-bis(pyridyloxy) benzene (TCPOBOP). Our results show that PXR was required for PCN-activation of Cyp2b2, Cyp3a23/3a1, Cyp3a18, Cyp2c11 and Slco1a2 (Oatp1a2) gene expression, and that CAR was required for TCPOBOP-activation of Cyp2b2 and Cyp3a23/3a1 gene expression, in good agreement with data obtained from in vivo rat knockout models, human primary hepatocytes, nuclear receptor knock-out cell lines and mouse knock-out models. Hepatocytes from the PXR and CAR knockout rats therefore represent a useful ex vivo complement to the in vivo models and tools for drug development, especially for functional studies on metabolic pathways involving nuclear receptors.