The fluorescence anisotropy of GFP was used to probe of the effect of high cellular biopolymer concentrations on rotational diffusion in E. coli. GFPmut2 expressed in the cytosol of E. coli K12 (MG1655) was studied using time-correlated single photon counting (TCSPC). The anisotropy of cytoplasmic GFP was found to decay as a single exponential with rotational correlation time of ϕ = 35 +/- 4 ns. The reduction factor for rotational diffusion of cytoplasmic GFP over GFP in buffer is Hr = D0/D rot ≈ 3. This is less than the reduction in translational diffusion (Ht ≈ 7) previously found for cytosolic GFPmut2 under similar cellular conditions. The rotational correlation time for GFP in solutions of increasing viscosity and solutions of increasing protein concentration were measured in order to quantify possible contributions of cellular viscosity and protein-protein interactions to Hr. The reduction in rotational diffusion could not be explained by either of these effects alone. We also describe the TCSPC microscope built to carry out these measurements, its use in collected TCSPC data, and a description of the software created for analysis of the TCSPC data.