Programmed cell death (apoptosis) refers to a specific type of cell death under stringent genetic control. Even a slight alteration in this process leads to malformations characterized by birth defects. Based on the above hypothesis we deduced that apoptosis plays an important role in mediating the teratogenicity of cyclophosphamide in vitro. The present study was undertaken to see whether this phenomenon holds true or not. In this study, 11-day-old rat embryos were cultured for 24 hours with various concentrations of CP (i.e. 0, 5, 10 and 100 mug/ml culture). After culturing for 24 hours, embryos exposed to 10 and 100 mug/mL culture of CP were found having both malformations and growth retardation. Exposure to CP at 5 mug/mL culture did not show significant effect on embryonic development. Parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were also carried out followed by quantitation by 3'-OH labeling of cultured embryos to evaluate CP-induced apoptosis. All the results were found to be dose-dependant, and the data suggested that apoptosis is involved in mediating the teratogenicity of CP in vitro.