BACKGROUND AND PURPOSE: One of the routinely used intraoperative tests for diagnosis of periprosthetic infection (PPI) is the Gram stain. It is not known if the result of this test can vary according to the type of joint affected or the number of specimen samples collected. We examined the role of this diagnostic test in a large cohort of patients from a single institution. MATERIALS AND METHODS: A positive gram stain was defined as the visualization of bacterial cells or "many neutrophils" (> 5 per high-power field) in the smear. The sensitivity, specificity, and predictive values of each individual diagnostic arm of Gram stain were determined. Combinations were performed in series, which required both tests to be positive to confirm infection, and also in parallel, which necessitated both tests to be negative to rule out infection. RESULTS: The presence of organisms and "many" neutrophils on a Gram smear had high specificity (98-100%) and positive predictive value (89-100%) in both THA and TKA. The sensitivities (30-50%) and negative predictive values (70-79%) of the 2 tests were low for both joint types. When the 2 tests were combined in series, the specificity and positive predictive value were absolute (100%). The sensitivity and the negative predictive value improved for both THA and TKA (43-64% and 82%, respectively). INTERPRETATION: Although the 2 diagnostic arms of Gram staining can be combined to achieve improved negative predictive value (82%), Gram stain continues to have little value in ruling out PPI. With the advances in the field of molecular biology, novel diagnostic modalities need to be designed that can replace these traditional and poor tests.