A sensitive and specific method has been developed for the simultaneous analysis of different O6-alkylguanines. The cross-reactivity of two different antibodies raised against O6-methylguanosine and O6-butylguanosine for a series of O6-alkylguanines was exploited for the immunoaffinity purification of biological samples before quantitative analysis by gas chromatography/mass spectrometry. The method can be applied to the detection of O6-alkylguanines in DNA and appears to be useful for studying chemical carcinogen mechanisms in animals and possibly for the detection of human exposure to alkylating agents.