Abstract— The excitation of pBR322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width ∼140 fs, 76 MHz pulse repetition rate, average power output focused through 10×microscope objective is ∼1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 μM of the intercalator proflavine (PF), both direct single-and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 ± 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/ cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 ± 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (∼400–500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400–500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single-and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.