In order to characterize the pharmacokinetics, excretion, and distribution of combretastatin A4 phosphate (CA4P) and its active metabolite, combretastatin A4 (CA4), in rats, a reliable gradient HPLC-based method has been developed and validated. The pharmacokinetic profiles of CA4P and CA4 in rats after CA4P intravenous injection at doses of 0.7, 1 and 4 mg x kg(-1) were best described by a two-compartment model. The terminal half-lives of CA4P or CA4 were similar at different CA4P dose levels, 5-9 min for CA4P and 39-60 min for CA4, while t1/2alpha, and Vd of CA4P or CA4 were very different. CA4 was largely distributed to the heart, intestine, lung, spleen and liver during 15 to 40 min after intravenous injection of CA4P. CA4P was predominantly excreted into urine (10.72%) and feces (9.703%) and to a lesser extent into bile (0.897%), whereas a greater portion of CA4 were excreted into feces (6.235%) and to a lesser extent into urine (0.782%) and bile (0.496%) during 0-28 h after intravenous injection of 1 mg x kg(-1) to rats. This is the first study to characterize the distribution of the active CA4P metabolite, CA4, in rat.