Monomethine cyanine dye 4-((1-methylbenzothiazolyliliden-2)methyl)-1,2,6-trimethylpyridinium perchlorate (Cyan 40) was investigated as a two-photon–excited fluorescence probe for nucleic acids (NA). Cyan 40 has been shown to demonstrate efficient two-photon–excited fluorescence in the presence of NA in vitro in contrast to solutions without NA. Two-photon confocal laser scanning microscopy (TPCLSM) and two-photon laser scanning microspectrofluorometry were used to check the possibility of using Cyan 40 as two-photon–excited fluorescence label for NA in living cells. Study of dye effect on viability of cells was also carried out. We ascertained that Cyan 40 is a cell-permeant dye, manifesting efficient two-photon–excited fluorescence when bound to NA in living cells, without any significant influence on viability of cells. TPCLSM images obtained from stained cells indicate preferential RNA staining by Cyan 40 compared with DNA.