Green fluorescent protein (GFP) was evaluated as a non-invasive probe to quantify the rheological properties and pH of intracellular aqueous compartments. Spectroscopic studies were done on several bacterially-expressed and purified GFP mutants. GFP-S65T was brightly fluorescent in solution (ƛex 492 nm, ƛem 509 nm) with lifetime 2.9 ns and rotational correlation time ( tc ) ∼20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 ± 2 ms (5 μm diameter spot) giving a diffusion coefficient of 8.7 x 10-7 cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein, GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), independent of spot diameter, and not affected by O2 or quenchers.