Acriflavine (ACF; CAS 8063-24-9), a mixture of trypaflavine (TRF) and proflavine (PRF) at a ratio of 2:1 is being investigated in rodents as an anticancer agent. However, its pharmacokinetics have not been investigated in mammals. Guanosine is known to potentiate the anticancer activity of some compounds. The pharmacokinetics of AG60, a 1:1 mixture of ACF and guanosine, were therefore investigated in rats. Rats were given 2 or 10 mg kg(-1) AG60 by intravenous bolus or 6 or 30 mg kg (-1) intramuscularly. An HPLC-based method was developed to analyse the levels of TRF, PRF, and their metabolites in plasma, bile, urine and tissue homogenates. The plasma concentrations of TRF and PRF decreased rapidly after intravenous administration and more slowly after intramuscular administration. Both TRF and PRF were distributed widely, most notably in the kidney, and were eliminated slowly. Three glucuronosyl conjugate metabolite peaks were tentatively identified in the bile. The intramuscular route leads to a prolongation of TRF or PRF plasma levels, and the systemic exposures for both TRF and PRF were both relatively high. These observations indicate that the intramuscular route may be the best way to administer AG60 for various clinical applications.