Arteriovenous fistulas are the preferred access type for hemodialysis. The buttonhole needling technique has become an alternative to stepladder or area puncture. However, an increased risk for infection has been described. The present study examined the risk for infectious complications with different needling techniques. Study Design: Prospective multicenter observational cohort study with 5 years of follow-up. Setting & Participants: In-center hemodialysis patients from 5 hemodialysis units in Denmark, dialyzed on a native arteriovenous fistula. 286 patients were included; 144 cannulated with the buttonhole technique. Exposure: The buttonhole cannulation technique was compared to the stepladder or area puncture technique. Outcomes: Primary end points: event rates of access-related Staphylococcus aureus bacteremia and the HR for first access-related S aureus bacteremia. Secondary end points: local infections and access-related S aureus bacteremia-related metastatic infections and mortality. Analytical Approach: Time-to-event analysis using Cox proportional hazards regression to estimate the HR of access-related S aureus bacteremia in buttonhole cannulation compared to stepladder/ area puncture. Poisson regression was used for incidence rate ratio calculations. Results: S aureus caused 48 access-related bacteremias; 43 (90%) in the buttonhole group compared with 5 (10%) in the stepladder/area group. The HR for first access-related S aureus bacteremia was significantly higher for buttonhole cannulation compared to stepladder/area needling (unadjusted, 6.8 [95% CI, 2.4-19.1]; adjusted, 8.4 [95% CI, 2.9-24.2]). The incidence rate ratio for access-related S aureus bacteremia was 6.8 (95% CI, 2.9-16.1), and the incidence rate ratio of local cannulation-site infection without access-related S aureus bacteremia was 3.8 (95% CI, 1.3-15.4) for buttonhole cannulation compared to stepladder/area needling. Limitations: Nonrandomized observational design, prevalent hemodialysis patients. Conclusions: Access-related S aureus bacteremia rates were very high for buttonhole cannulation compared to stepladder/area needling, questioning the use of buttonhole cannulation in routine clinical practice. A restrictive approach to buttonhole use is recommended, with buttonhole cannulation only being used as a second alternative to area technique when stepladder cannulation is not feasible. Complete author and article information provided before references.