Steady-state fluorescence anisotropy measurements can be used to detect fluorescence resonance energy transfer (FRET) between identical fluorophores (homo-FRET). However, the contribution of homo-FRET to the steady-state anisotropy must be discerned from those due to the orientational distribution and rotational diffusion, which so far has required photobleaching controls, largely precluding dynamic measurements in live cells. We describe a variation of steady-state anisotropy microscopy in which the contribution of homo-FRET is dynamically isolated from the total anisotropy by exploiting the loss of energy transfer that occurs at red-edge excitation. Excitation of enhanced green fluorescent protein (EGFP) at the red-edge of its absorption band shows the shift in the emission spectrum compared to main-band excitation that is characteristic for photo-selection of static low energy So-S, transitions that fail to exhibit FRET. An experimental setup for steady-state fluorescent anisotropy microscopy is described that can be used to acquire anisotropy images in live cells at main-band and red-edge excitation of EGFP. We demonstrate in live cells homo-FRET suppression of protein fusion constructs that consist of two and three EGFP molecules connected by short linkers. This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules. (C) 2003 Elsevier Inc. All rights reserved.