Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene. On incubation of the reacted DNA, apurinic sites developed which gave rise to strand breakage in alkaline solution. The reduction in molecular weight produced by these breaks was measured by analytical ultracentrifugation. In the case of anti-BPDE this depurination was shown to occur in two stages. The first was mainly due to attack at the 7-position of guanine, to yield an adduct which was lost from the DNA within a few hours. The second stage was due to much slower loss of the major N2-guanine adduct. The separated enantiomers, (+)- and (-)-anti-BPDE, and syn-BPDE all caused depurination to various extents. It is argued that although these processes are important in a study of the action of BPDE on DNA in vitro, their contribution to the biological activity of BPDE is probably negligible.