Background: The white blood-cell count and neutrophil differential of the synovial fluid have been reported to have high sensitivity and specificity in the diagnosis of periprosthetic infection following total knee arthroplasty. We hypothesized that neutrophils recruited into an infected joint secrete enzymes that may be used as markers for infection. In this prospective study, we determined the sensitivity and specificity of one of these enzymes, leukocyte esterase, in diagnosing periprosthetic joint infection. Methods: Between May 2007 and April 2010, synovial fluid was obtained preoperatively from the knees of patients with a possible joint infection and intraoperatively from the knees of patients undergoing revision knee arthroplasty. The aspirate was tested for the presence of leukocyte esterase with use of a simple colorimetric strip test. The color change (graded as negative, trace, +, or ++), which corresponded to the level of the enzyme, was noted after one or two minutes. Results: On the basis of clinical, serological, and operative criteria, thirty of the 108 knees undergoing revision arthroplasty were infected and seventy-eight were uninfected. When only a ++ reading was considered positive, the leukocyte esterase test was 80.6% sensitive (95% confidence interval [CI], 61.9% to 91.9%) and 100% specific (95% CI, 94.5% to 100.0%), with a positive predictive value of 100% (95% CI, 83.4% to 100.0%) and a negative predictive value of 93.3% (95% CI, 85.4% to 97.2%). The leukocyte esterase level correlated strongly with the percentage of polymorphonuclear leukocytes (r = 0.7769) and total white blood-cell count (r = 0.5024) in the aspirate as well as with the erythrocyte sedimentation rate (r = 0.6188) and C-reactive protein level (r = 0.4719) in the serum. Conclusions: The simple colorimetric strip test that detects the presence of leukocyte esterase in synovial fluid appears to be an extremely valuable addition to the physician's armamentarium for the diagnosis of periprosthetic joint infection. The leukocyte esterase reagent strip has the advantages of providing real-time results, being simple and inexpensive, and having the ability to both rule out and confirm periprosthetic joint infection. However, additional multicenter studies are required to substantiate the results of our preliminary investigation before the reagent strip can be used confidently in the clinic or intraoperative setting. Level of Evidence: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence. Copyright © 2011 by The Journal of Bone and Joint Surgery, Incorporated.