Release of DNA fragments into the culture supernatant of human promyelocytic cell line HL-60 was investigated. The released DNA has previously been shown to have a strong immunosuppressive effect on the proliferation of mitogen-stimulated human peripheral lymphocytes and some cancer cell lines. In this study, HL-60 cells were cultured for four days in serum-free medium and the amount of extracellular nucleic acid was monitored daily. Nucleic acids were isolated by phenol/chloroform extractions, followed by ethanol precipitations. A similar amount of DNA was identified in the HL-60 culture supernatant regardless of whether the cells were incubated for one day or up to four days. Also, the same amount of DNA was isolated from culture supernatants after washing the cells daily and reincubating into the fresh medium. During the first 8 hours of incubation no DNA material was detected in the culture supernatant, whereas at 24 h the concentration of the extracellular DNA reached a plateau. The data suggest the extracellular DNA is not accounted for totally by DNA from dying cells, and a regulatory mechanism may be involved which controls the release of DNA into the medium.