Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/CAD DNase. DFF40/CAD primary activated through site‐specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF‐7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF‐7. Activation and accumulation of 40–50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate‐treated MCF‐7 were detected by SDS‐DNA‐PAGE assay. Microsomes of apoptotic MCF‐7 activate 40–50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac‐DEVD‐CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF‐7.