Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible.