Aziridinyl substituted cyclophosphazenes are a new group of inorganic chemical agents with in vitro and in vivo cytotoxic activity. We investigated the mode of action on DNA of three different compounds, 1,3,3,5,5-pentakis (1-aziridinyl)-1 lambda 6,2,4,6,3 lambda 5,5 lambda 5-thiatriazadiphosphorine -1-oxide (SOAz), trans-1,3-bis(1-aziridinyl)-1,3,5,5-tetrakis (methylamino)-2,4,6,1 lambda 5,3 lambda 5,5 lambda 5-triazatriphosphorine (AZP), and 1,trans-5-bis(1-azaridinyl)-gem-1,3,3'-cis-5,7,7'-hexakis (methylamino)-2,4,6,8,1 lambda 5,3 lambda 5,5 lambda 5,7 lambda 5 -tetraazatetraphosophocine (AZM), of this group in the Ehrlich ascites tumor cell line (EAT) and a human small cell carcinoma cell line. The DNA damage was evaluated by alkaline elution and ethidium bromide fluorescence assay. Each compound gave a different pattern of DNA damage. SOAz caused neither single strand breaks nor cross-links, AZP gave cross-links, and AZM gave single strand breaks and cross-links in both cell lines after drug incubation for 6 h. The range of concentrations leading to cytoxicity of AZP and AZM in the clonogenic assay coincided with the concentrations leading to DNA damage. Cell kill occurred with SOAz in the same range of concentrations, however, without detectable evidence of DNA damage. It was concluded that cyclophosphazenes are probably a heterogeneous group as far as their mode of action as cytostatic agents is concerned.