The fluorescence intensity and lifetime of the 4,4'-difluoro-4-bora-5-(p-oxoalkyl)phenyl-3a,4a-diaza-s-indacene (1) show a strong correlation with the viscosity of the medium due to the viscosity-dependent twisting of the 5-phenyl group, which gives access to the dark nonemissive excited state. We propose a sensitive and versatile method for measuring the local microviscosity in biological systems, based on the determination of the fluorescence lifetime of 1. Fluorescence lifetime imaging (FLIM) performed on live cells incubated with 1 demonstrates the distinct intracellular lifetime of the molecular rotor of 1.6 +/- 0.2 ns corresponding to the intracellular viscosity of ca. 140 cP. Time-resolved fluorescence anisotropy of 1 in cells confirms insignificant binding of the fluorophore. The viscosity value obtained in the present study is considerably higher than that of water and of cellular cytoplasm. The high viscosity of intracellular compartments is likely to play an important role in vital intracellular processes, including the rate of diffusion of reactive oxygen species, causing programmed cell destruction.