Our group has recently identified, in human semen, round bodies of different size and density which were termed M540 bodies due to their staining with the fluorochrome merocyanine 540. Here, we investigate the hypothesis that such structures represent apoptotic bodies. To this aim, by both fluorescence-activated cell sorting (FACS) and fluorescence microscopy, we examined the occurrence of apoptotic markers such as caspase activity, Fas, p53 and Bcl-x in M540 bodies. In addition, we evaluated their ultrastructure by transmission electron microscopy. We found that M540 bodies express all the investigated markers, strongly supporting our hypothesis. We also found that M540 bodies contain fragmented DNA, another evidence of their apoptotic derivation. We investigated also the presence of M540 bodies in the different categories of patients. With respect to normozoospermic subjects, a higher content of M540 bodies was found in oligoasthenoteratozoospermic and asthenoteratozoospermic, but not in asthenozoospermic and teratozoospermic men. Interestingly, these subjects are those whose semen shows the highest levels of apoptotic signs. The variable occurrence of apoptotic bodies in semen may thus be considered a sign of abortive apoptosis in male reproductive organs. Of interest, since M540 bodies exhibit a similar size and density to sperm, they represent a confounding factor in FACS studies on ejaculated sperm.