Trophoblast injury may be one of the possible causes of fetal distress associated with chemotherapy administered during pregnancy. The purpose of this study was to investigate theex vivochemosensitivity of normal trophoblasts (NTB) against commonly used antineoplastic agents. Using the newly developedex vivoATP-based trophoblast assay (ATP-TBA), 31 NTB freshly sampled from human placentas (gestational week 7–42) were tested against dactinomycin (Act-D), 5-fluorouracil (5-FU), 4-OOH-cyclophosphamide (4-HC), vincristine (VCR) and methotrexate (MTX) alone or in combination with calcium folate (LV). All agents were studied at concentrations relevant to clinical dosages normally used for chemotherapy of solid neoplasms. Of 31 samples studied with the ATP-TBA, 20 (65%) were evaluable. VCR, Act-D and 4-HC were the most active drugs with 55, 45 and 45% of samples respondingex vivo. Antimetabolites were less active, producingex vivoresponse rates of 25 (MTX) and 20% (5-FU), respectively. MTX activity was largely neutralized by adding LV. The chemosensitivity of NTB showed considerable inter-individual variations and did not decrease with increasing gestational age. We therefore conclude that NTB of any gestational age exhibit considerableex vivosensitivity against common anticancer agents which is comparable to that observed for various solid tumors. The ATP-TBA may be helpful in planning future trials with both single agents and drug combinations in order to standardize and optimize chemotherapy during pregnancy.