Preclinical studies are currently underway to examine the potential antitumor effects of a 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine. Guanosine potentiates the anti-cancer activity of some compounds. However, the effects of guanosine on the pharmacokinetics of ACF in mammals are unknown. Therefore, this study investigated the effects of guanosine on the pharmacokinetics of ACF after administering a 1:1 mixture of ACF and guanosine in rats. The rats were given either 10 mg/kg of the mixture or 5 mg/kg ACF via an intravenous bolus injection; or 30 mg/kg of the mixture or 15 mg/kg ACF intramuscularly. An HPLC-based method, which was validated in this laboratory, was used to analyze the levels of trypaflavine (TRF) and proflavine (PRF) in the plasma, bile, urine, and tissue homogenates. It was found that TRF and PRF were rapidly cleared from the blood and transferred to the tissues after the i.v. bolus or i.m. injection of the combination mixture. Both TRF and PRF were found to be most highly concentrated in the kidneys after the i.v. bolus or i.m. injection, followed by slow excretion to the bile or urine. Guanosine had no effect on the plasma disappearance of TRF or PRF after the i.v. bolus injection. However, guanosine led to a prolongation of the plasma levels of PRF after the i.m. administration of the combination mixture, resulting in a 2 fold increase in the bioavailability (BA) of PRF. The concentrations of TRF and PRF in all the tissues examined were similar in the groups given the mixture and ACF. However, guanosine led to a prolongation of the biliary and urinary excretions of both TRF and PRF after the i.v. bolus (1.25 fold) or i.m. (1.5-2.4 folds) injection. These prolonged effects of guanosine on the plasma disappearance or urinary excretion of TRF and PRF might be one reason for the enhanced antitumor effects of ACF. However, more study will be needed to further examine this potential mechanism.