In order to study the effect of phenobarbital(PB) on the hepatic transport of diltiazem(DTZ), $Ca^{2+}$ channel blocker, we used isolated hepatocytes of rat which was intraperitoneally pretreated with phenobarbital sodium(75 mg/kg) for four days once a day. For the isolation of rat liver cells, a modification of the two step procedure of Seglen was used. DTZ was dissolved in incubation buffer to the final DTZ concentrations of 200, 400, 600, 800 and 1000 ng/ml in order to elucidate the uptake characteristics of DTZ by hepatocytes. Reactions were stopped at 10, 20, 30, 45, 60, 90, 120 and 300 sec. The initial velocity was determined by disappearance of diltiazem in the hepatocyte suspension. On the other hand, to determine the effect of PB on the in vitro hepatic intrinsic clearance of DTZ we obtained the metabolism rates of DTZ in the control and the PB-pretreated rat hepatocyte at various time intervals. According to pretreatment with PB, the size of hepatocyte and the amount of protein per $10^6$ cells were significantly (p<0.01) increased from $26.92{pm}0.1364;m$ to $35.31{pm}1.00;m$ and from $468{pm}6.5;{mu}g/10^6$ cells to $628.8{pm}12.1{mu}g/10^6$ cells, respectively. In the case or hepatic uptake of diltiazem, $K_m$ was not different in the normalization by cell numbers and increased from $2.90;{mu}M;to;13.89;{mu}M$ in the normalization by protein amount. $V_max$ was increased regardless of normalization by protein amount and cell numbers, from $1.21;{mu}mole/min ;{cdot};mg;protein;to;3.96;{mu}mole/min;{cdot};mg;protein;and;from;2.38;{mu}mole/min;{cdot};10^6;cells;to;2.83;{mu}mole/min;{cdot};10^6;cells$, respectively. The in vitro hepatic intrinsic clearance of DTZ was significantly (p<0.01) increased from $0.640{pm}0.038;ml/mim;{cdot};10^6;cells;to;2.385{pm}0.212;ml/min;{cdot};10^6;cells$ due to PB-pretreatment. These results suggest that the uptake of DTZ by hepatocyte is extremely fast and PB enhances the hepatic intrinsic metabolic clearance of DTZ.