OBJECTIVE: Conjugation of arginine-rich cell-penetrating peptide (CPP) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance cytosolic and nuclear delivery of PMO. However, the in vivo disposition of CPP-PMO is largely unknown. In this study, we investigated the pharmacokinetics, tissue distribution, stability, and safety profile of an anti-c-myc PMO conjugated to the CPP, (RXR)4 (X = 6-aminohexanoic acid) in rats. METHODS: The PMO and CPP-PMO were administrated intravenously into rats. The concentrations of the PMO and the CPP-PMO in plasma and tissues were monitored by HPLC. The stability of the CPP portion of the CPP-PMO conjugate in rat plasma and tissue lysates was determined by mass spectrometry. The safety profile of the CPP-PMO was assessed by body weight changes, serum chemistry, and animal behavior. RESULTS: CPP conjugation improved the kinetic behavior of PMO with a 2-fold increase in the estimated elimination half-life, a 4-fold increase in volume of distribution, and increased area under the plasma concentration vs time curve. Consistent with the improved pharmacokinetic profile, conjugation to CPP increased the uptake of PMO in all tissues except brain, varied between organ type with greater uptake enhancement occurring in liver, spleen, and lungs. The CPP-PMO conjugate had greater tissue retention than the corresponding PMO. Mass spectrometry data indicated no observable degradation of the PMO portion, while there was identifiable degradation of the CPP portion. Time-dependent CPP degradation was observed in plasma and tissue lysates, with the degradation in plasma being more rapid. The pattern of degraded products differed between the plasma and lysates. Safety evaluation data showed that the CPP-PMO was well-tolerated at the dose of 15 mg/kg with no apparent signs of toxicity. In contrast, at the dose of 150 mg/kg, adverse events such as lethargy, weight loss, and elevated BUN (p < 0.01) and serum creatinine (p < 0.001) levels were recorded. Supplementation with free L-arginine ad libitum showed improved clearance of serum creatinine (p < 0.05) and BUN (p < 0.01) at the toxicological dose, suggesting that the CPP caused toxicity in kidney. CONCLUSION: This study demonstrates that conjugation of CPP to PMO enhances the PMO pharmacokinetic profile, tissue uptake, and subsequent retention. Therefore, when dosed at < or = 15 mg/kg, CPP is a promising transporter for enhancing PMO delivery in therapeutic settings.