The alpha-diazo ketones 1, 10, and 11 were designed as mimics for the radical-generating system of neocarzinostatin chromophore (7). These alpha-diazo ketones are able to generate diradicals under thermal or photoirradiation conditions in toluene via the cyclization of the ene-yne-ketene intermediates. Ab initio MO calculations revealed that the efficiency of the radical generation is highly dependent on the conformation of the alpha-diazo ketones which is controlled by the substituent on the carbon directly attached to the diazo group. Two alpha-diazo ketones 10 and 11 having a DNA binding group considerably improved the DNA-cleaving activity compared to that for 1 under photoirradiation at 366 nm. The absence of an appreciable amount of cyclized indanol 23 in the photoirradiated solution of 10 in the presence of pBR322 DNA strongly suggests that the diradical is not responsible for the observed DNA cleavage. Likewise, photoirradiation of 1 in a 50% aqueous acetonitrile solution did not produce indanol 5 but gave a novel furan derivative 33. The increased yield of 33 under aerobic conditions suggested that the mechanism producing 33 involves the trapping of photogenerated alpha-keto carbene 29 with molecular oxygen. These experiments together with theoretical calculations indicated that alpha-keto carbenes generated by photoirradiation of alpha-diazo ketones may be the principal DNA-cleaving species.