Irinotecan suppository was prepared using the moulding method with a homogeneous blend. A sensitive and specific fluorescence method was developed and validated for the determination of irinotecan in plasma using HPLC. The pharmacokinetics of intravenous administered and rectal administered in rabbits was investigated. Following a single intravenous dose of irinotecan (50 mg/kg), the plasma irinotecan concentration demonstrated a bi-exponential decay, with a rapid decline over 15 min. $C_{max}$, $t_{1/2}$, $AUC_{0-30h}$ and $AUC_{0-{infty}}$ were $16.1{pm}2.7g/ml$, $7.6{pm}1.2h$, $71.3{pm}8.8{mu}g{cdot}h/ml$ and $82.3{pm}9.5{mu}g{cdot}h/ml$, respectively. Following rectal administration of 100 mg/kg irinotecan, the plasma irinotecan concentration reached a peak of $5.3{pm}2.5{mu}g/ml$ at 4 h. The $AUC_{0-30h}$ and $AUC_{0-{infty}}$ were $32.2{pm}6.2{mu}g{cdot}h/ml$ and $41.6{pm}7.2{mu}g{cdot}h/ml$, respectively. It representing ~50.6% of the absolute bioavailability.