Background: Airborne infection isolation rooms (AIIRs) help prevent the spread of infectious agents in hospitals. The performance of 678 AIIRs was evaluated and compared with construction design guidelines. Methods: The pressure differentials (ΔP) between the isolation rooms and adjacent areas were measured, and ventilation and construction details were recorded for each room. Ultrafine particle concentrations were evaluated in the rooms, surrounding areas, and ventilation systems serving the rooms. Measurements were analyzed as a function of room parameters. Results: Only 32% of the isolation rooms achieved the recommended ΔP of -2.5 Pascals (Pa) relative to surrounding areas. AIIRs with solid ceilings had an average ΔP of -4.4 Pa, which was significantly higher than the average ΔP of -2.0 Pa for rooms with dropped ceilings (P = .0002). Isolation room ultrafine particle concentrations were more highly correlated with particle levels in surrounding areas (R2 = 0.817) than in the ventilation systems serving the rooms (R2 = 0.441). Almost all ventilation filters serving AIIRs collected fewer particles than anticipated. Conclusion: The results indicate that hospitals are not all maintaining AIIRs to correspond with current guidelines. The findings also support the contention that having tightly sealed rooms helps maintain appropriate pressure differentials.