Background: There is increasing interest in Positron Emission Tomography (PET) of 2-deoxy-2-[18F]flouro-D-glucose (18F-FDG) to evaluate pulmonary inflammation during acute lung injury (ALI). We assessed the effect of extra-vascular lung water on estimates of 18F-FDG-kinetics parameters in experimental and simulated data using the Patlak and Sokoloff methods, and our recently proposed four-compartment model. Methodology/Principal Findings: Eleven sheep underwent unilateral lung lavage and 4 h mechanical ventilation. Five sheep received intravenous endotoxin (10 ng/kg/min). Dynamic 18F-FDG PET was performed at the end of the 4 h period. 18F-FDG net uptake rate (Ki), phosphorylation rate (k3), and volume of distribution (Fe) were estimated in three isogravitational regions for each method. Simulations of normal and ALI 18F-FDG-kinetics were conducted to study the dependence of estimated parameters on the transport rate constants to (k5) and from (k6) the extra-vascular extra-cellular compartment. The four-compartment model described 85.7% of the studied 18F-FDG-kinetics better than the Sokoloff model. Relative to the four-compartment model the Sokoloff model exhibited a consistent positive bias in Ki (3.32 [1.30-5.65] 10-4/min, p<0.001) and showed inaccurate estimates of the parameters composing Ki (k3 and Fe), even when Ki was similar for those methods. In simulations, errors in estimates of Ki due to the extra-vascular extra-cellular compartment depended on both k5 and k5/k6, with errors for the Patlak and Sokoloff methods of 0.02 [-0.01-0.18] and 0.40 [0.18-0.60] 10-3/min for normal lungs and of -0.47 [-0.89-0.72] and 2.35 [0.85-3.68] 10-3/min in ALI. Conclusions/Significance: 18F-FDG accumulation in lung extra-vascular fluid, which is commonly increased during lung injury, can result in substantial estimation errors using the traditional Patlak and Sokoloff methods. These errors depend on the extra-vascular extra-cellular compartment volume and its transport rates with other compartments. The four-compartment model provides more accurate quantification of 18F-FDG-kinetics than those methods in the presence of increased extra-vascular fluid.