N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) is an alkylating agent that forms DNA adducts at C-8 in guanine and causes single strand breaks. It has previously been shown to be embryotoxic, but the mechanisms by which it causes abnormal development have not been investigated. Previous studies have indicated that other DNA alkylating agents cause cell death during embryonic development although the types of cell death were not characterized. Using a whole embryo culture system, gestation day 10 rat embryos were exposed to several concentrations (5, 50, and 200 micrograms/ml) of N-Ac-AAF. At several time points after exposure was begun (5, 10, and 24 hours), the embryos were removed from culture and examined to identify location, type and quantity of cell death, relative to programmed cell death observed in control embryos. Vital staining with Nile blue sulphate revealed that the location of N-Ac-AAF-induced cell death included the forebrain region, tail, and areas of programmed cell death. Examination of tissue sections from both control and treated embryos indicated that the location of apoptotic cell death revealed by in situ DNA nick end-labelling was generally consistent with the cell death pattern observed by vital staining of whole embryos. Agarose gel analyses indicated that all concentrations of N-Ac-AAF caused DNA fragmentation, and quantification demonstrated a dose response. Examination of treated embryos (50 and 200 micrograms/ml) by transmission electron microscopy revealed that, by 5 hours after exposure, cells with classic, ultrastructural features of apoptosis were present. In conclusion, multiple methods have all indicated that, regardless of exposure level, apoptosis was the predominant form of cell death. Because apoptosis also occurs in developmental cell death, it is possible that apoptosis induced by N-Ac-AAF is due to an alteration in cell fate via premature or ectopic induction of the cell death program.