The sequence dependency of the antitumor effect of etoposide and cytarabine (ara-C) was investigated against the L1210 ascites tumor in BDF1 mice. Etoposide (7.5 mg/kg or 15 mg/kg) and ara-C (25 mg/kg or 500 mg/kg) were administered intraperitoneally on days 1, 4, and 7 after inoculation of L1210 cells with or without a time interval of 3 or 6 h. Simultaneous administration of etoposide and ara-C produced a 70% cure rate. At every dosage examined, pretreatment with etoposide given 6 h before ara-C was the most effective antitumor schedule in L1210 leukemia. At 1 h after injection of ara-C, 3 h and 6 h pretreatment with etoposide 15 mg/kg increased ara-C incorporation to more than 200% as compared with that of ara-C given alone. Simultaneous administration of etoposide, however, decreased ara-C incorporation to 33% of that of ara-C alone. Deoxycytidine kinase (dCK) is a rate-limiting enzyme for the activation of ara-C. We demonstrated that dCK activity was increased within 1 h after exposure to etoposide. Much more attention must be paid to the timing of the administration of etoposide in combination chemotherapy with etoposide and ara-C.