Background: For critically ill adults receiving empirical vancomycin, the duration of negative cultures after which vancomycin may be discontinued without risking subsequent growth of methicillin-resistant Staphylococcus aureus (MRSA) remains unknown. Objective: We hypothesized that if sputum cultures did not grow MRSA or blood cultures did not grow Gram-positive cocci on Gram stain by 48 hours, those cultures would not subsequently demonstrate MRSA. Methods: We conducted an ancillary analysis from patients enrolled in the Isotonic Solutions and Major Adverse Renal Events Trial (SMART). In this cohort of patients, we collected data on the time of either MRSA identification in culture or Gram-positive cocci identification on Gram stain and rate of vancomycin discontinuation. Results: Of the 15 802 patient admissions in the SMART study, 6553 (41.5%) received empirical intravenous vancomycin. Respiratory sputum cultures demonstrated MRSA during 178 patient admissions. Among respiratory cultures that would ultimately grow MRSA, 85% were positive within 48 hours, and 97% were positive within 72 hours. Cultures demonstrated MRSA bacteremia during 85 patient admissions. In 83 cases (97.6%) of MRSA bacteremia, Gram-positive cocci were identified within 48 hours after the culture was obtained. Conclusion and Relevance: This analysis of a large cohort of critically ill adults receiving empirical vancomycin found that Staphylococcus aureus was present in all but 15% of cases of MRSA-positive respiratory cultures after 48 hours, whereas Gram-positive cocci were identified within 48 hours during nearly all episodes of MRSA bacteremia. These findings may inform the timing of discontinuation of empirical vancomycin among critically ill adults.